Dietary immune-modulation – carbohydrate specific effects of cereal and yeast �-glucans
Philosophiae Doctor (PhD) Thesis
Anne Rieder
2013
Department of Nutrition
Institute of Basic Medical Sciences
Faculty of Medicine
University of Oslo
Nofima, Norwegian Institute of
Food, Fisheries and Aquaculture
Research
© Anne Rieder, 2013 Series of dissertations submitted to the Faculty of Medicine, University of Oslo No. 1521 ISBN 978-82-8264-525-6 All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission. Cover: Inger Sandved Anfinsen. Printed in Norway: AIT Oslo AS. Produced in co-operation with Akademika publishing. The thesis is produced by Akademika publishing merely in connection with the thesis defence. Kindly direct all inquiries regarding the thesis to the copyright holder or the unit which grants the doctorate.
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Acknowledgements
The work presented in this thesis was carried out at Nofima, the Norwegian Institute of Food, Fisheries and Aquaculture Research in Ås. The financial support was provided by the Fund for the Research Levy on Agricultural Products.
I would like to thank my supervisors at Nofima, Svein Halvor Knutsen and Stine Grimmer for their great support. You have been fantastic and I have learned so much from you in the last years! I could fill pages (you know I like looong paragraphs and loooong sentences) with praise, but will restrict myself to saying thanks for making me a part of the research team at Nofima.
I would also like to thank my supervisor at the University of Oslo, Svein Olav Kolset, for great scientific discussions, good ideas, the opportunities to present our work for his research group and of course also for the charming end-of-term celebrations at his house. Even before I started my PhD studies, I was trying to sing Norwegian Christmas songs at your house!
I want to thank all my co-authors for their contribution and expertise that greatly improved the quality of the presented scientific papers. A special thanks to Anne Berit Samuelsen at the Department of Pharmaceutical Chemistry at the University of Oslo for our joint project of immerging deeply into the world of cereal �-glucans. I really enjoyed working with you! And of course I would like to thank Diego for all the Saturdays we spent with the Fluorescence instrument until our method finally worked. I learned a lot form you and I miss our bike trips to Drøbak and your addiction to the day light lamp.
Merete, Signe, Elin, Linda, Helle, Hanne, Marte, Janne and Stine, I would like to thank you for sharing your knowledge of cell line cultures and all cell related test systems at Nofima with me. It was good to have someone to share the trouble with the “wee” cells with.
I want to thank all my colleagues at the dietary fiber research group for fruitful scientific discussions, a great working environment and all the cakes from the bakery. A special thanks to Ann Katrin for always being there for me at work and after work!
Thanks to my fellow PhD students and friends at Nofima and FODOS for all our social events and sharing our PhD frustration with each other. I would like to thank my office made Anastasia for nice conversations and friendly company. I thank Ulrike for her friendship and sharing the complete language confusion with me. Thanks also to Jib, Elena, Silje, Gunna, Natasa, Trygve, Johannes, Nebojsa, Håkon and Sarin for their company and friendship, all ski trips, cabin trips and day light lamp parties.
I want to thank my family and friends in Germany for their support and their frequent visits to Norway. What would I have done without all the phone calls complaining about the weather? �
I thank Nils for all our scientific discussions at the dinner table and especially for repeating “det kommer til å gå så bra” so many times that even I finally believed it. Let`s hope it`s true….min løk �
Ås, January 2013 Anne Rieder
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Table of Contents Abbreviations ............................................................................................................................. 5
Explanation of key expressions .................................................................................................. 7
1. Introduction ............................................................................................................................ 9
2. The immune system ............................................................................................................. 10
2.1 Innate and adaptive immune responses .......................................................................... 10
2.2 Cytokines ........................................................................................................................ 13
2.2.1 IL-8 (CXCL-8) ......................................................................................................... 13
2.2.2 TNF-� ....................................................................................................................... 14
2.2.3 IL-1� ........................................................................................................................ 15
2.3 The gut immune system .................................................................................................. 16
3. �-glucan ................................................................................................................................ 18
3.1 �-glucan structure and structure-function relationships ................................................. 18
3.2 �-glucan receptors ........................................................................................................... 23
3.3 Possible mechanisms behind immune modulation by dietary �-glucans ....................... 24
4. LPS ....................................................................................................................................... 27
5. Aims of the study ................................................................................................................. 31
6. List of papers and manuscripts ............................................................................................. 32
7. Results .................................................................................................................................. 33
7.1 Review of cereal �-glucans immune-modulating properties (Paper I) ........................... 33
7.2 Effect of high MW �-glucan fractions and arabinoxylan fractions isolated from barley on IL-8 secretion by the intestinal epithelial cell lines Caco-2 and HT-29, NF-�B activity in monocytic U937 cells and complement fixation (Paper II) .................................................. 33
7.3 Effect of cereal �-glucan fractions with different weight average MW (Mw) on the cytokine secretion by intestinal epithelial cell lines Caco-2 and HT-29 (Paper III) ............ 34
7.4 Development of a new method to measure low concentrations of cereal �-glucans in cell culture supernatants and application to analysis of cereal �-glucan transport over intact Caco-2 cell monolayers (Paper IV) ...................................................................................... 35
7.5 Tools to study the carbohydrate specific effect of cereal and yeast �-glucan preparations (Paper V) ............................................................................................................................... 37
7.6 Effect of cereal and yeast �-glucan preparations on THP-1 derived human macrophages, differentiated Caco-2 cells and Caco-2 macrophage co-cultures (Paper VI) ....................... 38
8. Discussion ............................................................................................................................ 39
8.1 Interaction with IEC ....................................................................................................... 39
8.2 Uptake ............................................................................................................................. 41
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8.3 Macrophages ................................................................................................................... 45
8.4 Carbohydrate specific effects ......................................................................................... 48
8.5 Beta-glucans structure-function relationship .................................................................. 52
8.6 Current knowledge of dietary �-glucans mechanisms of action ..................................... 53
9. Concluding Remarks ............................................................................................................ 56
10. Reference List .................................................................................................................... 58
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Abbreviations
AP-1 activator protein 1
CR3 complement receptor 3
DB degree of branching
DC dendritic cell
DP degree of polymerization
ELK-1 Eph-related tyrosine kinase
GI-tract gastrointestinal tract
IEC intestinal epithelial cell
IEL intraepithelial lymphocyte
Ig immunoglobulin
IL interleukin
LAL limulus amoebocyte lysate
LBP lipopolysaccharide binding protein
LPS lipopolysaccharide
MBL mannose binding lectin
M cell microfold cell
MW molecular weight
Mw weight average molecular weight
MyD88 myeloid differentiation primary response gene 88
NF-�B nuclear factor kappa-light-chain-enhancer of activated B cells
NK cells natural killer cells
NMR Nuclear magnetic resonance
PAMP pathogen associated molecular pattern
PBMC peripheral blood mononuclear cells
PRR pattern recognition receptor
6
ROS reactive oxygen species
SCFA short chain fatty acids
TEER trans-epithelial electrical resistance
TNF-� tumor necrosis factor alpha
TLR toll like receptor
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Explanation of key expressions
Dietary �-glucans
The expression dietary �-glucans is used in this thesis for all �-glucans that are consumed as a
part of the diet. This can be �-glucans that naturally occur in the human diet like cereal �-
glucans or �-glucans from baker's yeast or it can be �-glucans that are added to foods during
manufacturing as a functional food ingredient. In animal studies dietary �-glucans is used for
orally applied �-glucans.
Targeted enzymatic degradation
Targeted enzymatic degradation describes a technique that was developed as a part of this
thesis and can be used to determine the carbohydrate specific effect of e.g. a �-glucan
preparation. The technique is based on the degradation of the component of interest (here �-
glucans) by specific enzymes. Comparison of the activity of the degraded preparation with the
activity elicited by the un-degraded preparation makes it possible to draw conclusion about
the specific activity of the components of interest. In the case of �-glucans the complete
degradation of �-glucans in the test samples should result in the complete abrogation of
observed effects if the activity is actually mediated by the �-glucan molecules.
Immune competent cells
In this thesis the term immune competent cell is used for any cell type that may play a role in
immune responses. This includes also cells that are not part of the immune system like
intestinal epithelial cells since they play an important role in orchestrating gut immune
responses.
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1. Introduction
The consumption of dietary fiber is generally regarded as important for the maintenance of
human health. Dietary recommendations promote an increase in fiber consumption. A high
intake of dietary fiber is regarded as an important part of primary prevention of major diseases
such as type II diabetes, cardiovascular disease and several forms of cancer [1]. Since the
concept of dietary fiber was introduced in the 1970s, it`s definition has been constantly under
debate. In 2009, the Codex Alimentarius Commission adopted an internationally accepted
regulatory definition for dietary fiber: Carbohydrate polymers that are resistant to digestive
enzymes in the small intestine of humans [2]. These can be either naturally occurring in food
as consumed, obtained from food raw material or be synthetic carbohydrate polymers [2]. The
latter two groups need to have demonstrated physiological benefits to human health in order
to be defined as dietary fiber [2]. The classic documented physiological benefits of dietary
fiber consumption include increased laxation, decreased risk of cardiovascular disease and
type II diabetes and positive effects on weight management [3, 4]. Furthermore, a high fiber
intake, especially cereal fiber and whole grain products, has been linked to a reduced risk of
colorectal cancer [5]. This protective effect may be due to decreased intestinal transit time,
decreasing the exposure time to environmental or food carcinogens, and colonic fermentation
of fiber leading to butyrate production, which has a beneficial effect on colonocyte cell cycle
regulation [6, 7]. In recent years, dietary fiber has also been linked to immune-modulation [8].
Either as a part of the prebiotic effects or due to structure mediated direct interaction with
immune competent cells [9-11].
�eta-glucans are major cell wall components in some cereal grains and fungi, and form a
heterogeneous group of indigestible carbohydrates with possible immune-modulating
properties. Furthermore, cereal �-glucans are known for their ability to decrease blood
cholesterol levels, a risk factor for cardiovascular disease, mainly by increasing the viscosity
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in the gastrointestinal (GI) tract [12, 13], and the European Food Safety Authority has
recently approved a health claim on oat �-glucan's ability to reduce blood cholesterol [14].
�eta-glucans from different sources have shown potential for direct interaction with immune-
competent cells [15]. Potential benefits of dietary immune stimulation with �-glucans include
increased resistance to infection [9, 16], therapeutic effects in treatment of various cancers
[17-21] and dampening of allergic disease [22]. Research on the immune-modulating
properties of �-glucans has been focused on �-glucans from fungi. However, also cereal
derived �-glucans have shown beneficial effects both in vitro and in animal studies [23].
Despite the identification of several �-glucan receptors, the precise mechanisms of action of
the immune-modulating properties of dietary �-glucans (cereal or fungal) are still far from
understood. The aim of this thesis was therefore to increase the current understanding of how
dietary �-glucans may modulate the human immune system by focusing on the first step from
the gut lumen to the activation of effector cells. Since cereal �-glucans are a natural part of the
human diet they received the main focus, but also �-glucans from yeast were investigated for
comparison purposes.
2. The immune system
The immune system is very complex, and thus this paragraph gives a brief overview of the
most important modes of action relevant for studies on �-glucans.
2.1 Innate and adaptive immune responses
Beta-glucans have been shown to modulate both innate and adaptive immune responses [24,
25]. The innate immune system is the evolutionary older part of the immune system that can
react fast to a broad range of pathogens. The adaptive immune system on the other hand
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creates highly specific, very powerful responses to particular pathogens that often need some
days to fully develop.
Immune responses in the innate immune system are initiated by binding of so called pathogen
associated molecular patterns (PAMP) on pathogens to pattern recognition receptors (PRR)
such as toll like receptors (TLR). PAMPs are molecular structures that are shared by different
pathogen groups such as lipopolysaccharides (LPS) from gram negative bacteria (interacting
with TLR-4), or peptidoglycan from gram positive bacteria (binding to TLR-2). Also �-
glucans are PAMPs, even though only a small part of this group derives from pathogens such
as the opportunistic pathogenic yeast Candida albicans. PRR signaling may activate
phagocytosis or lead to the secretion of cytokines that recruit or activate other effector cells.
Figure 1 illustrates the effector cells of the innate and adaptive immune system. Besides
effector cells such as neutrophil, macrophages and NK cells, the innate immune system also
comprises a series of proteins that activate one another, called the complement system.
Opsonization, recruitment of phagocytes and assembly of the membrane attack complex are
the main effector functions of the complement system. The complement cascade can be
activated by three different pathways. The classical pathway is activated by antigen:antibody
complexes. The lectin pathway is initiated by the binding of carbohydrate binding proteins
such as mannose binding lectin (MBL) to specific carbohydrates on pathogen surfaces. The
alternative pathway is activated by the direct binding of activated complement component 3 to
the pathogen surface.
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Figure 1: Cells of the innate and adaptive immune system from [26]
The effector cells of the adaptive immune system are highly specific towards one specific
antigen and have to be activated by an antigen presenting cell that has encountered their
specific antigen in order to be able to exert their effector functions. The effector cells of the
adaptive immune system can be divided into T-cells, exerting cell mediated immunity, and B-
cells, producing antibody for humoral immunity. Beta-glucans have been shown to play a role
in T-cell activation and T-cell differentiation into different effector cell subsets [21]. T-cells
can be divided into CD8+ T-cells, which have cytotoxic effector functions and CD4+ T-cells,
whose effector function is to activate other cells like macrophages or B-cells. CD4+ T-cells
are therefore also called helper T-cells (TH). TH cells can develop into different subsets with
distinct functions depending on the cytokine environment. TH1 cells for example secret
macrophage activating molecules, while TH2 cells activate B-cells. TH17 cells are mainly
known for their ability to recruit neutrophils and are often found in the gut. Regulatory T-cells
(Treg) secrete suppressive cytokines such as TGF-� and IL-10 and play an important role in
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maintaining homeostasis e.g. in the gut. B-cells need T-cell help for their activation and the
cytokine environment created by T-cells also determines the immunoglobulin (Ig) class of the
antibody produced. The specific effector functions of the antibody (neutralization,
opsonization, complement and cell activation) depend on it's Ig class.
2.2 Cytokines
Cytokines are small, soluble secreted proteins or peptides that effect the growth or function of
cells. Mostly cytokines act in an autocrine (on the same cell that produces them) or paracrine
(cells in the near proximity) way [27]. However, TNF-�, IL-1� and IL-6 may also exhibit
systemic effects [28]. Even though cytokines may be secreted by somatic cells and somatic
cells may be affected by cytokines, their action usually comprises immune cells and they are
functionally involved in orchestrating most aspects of the immune system.
2.2.1 IL-8 (CXCL-8)
Cytokines that exert a chemotactic function towards leukocytes are called chemokines [29].
The most prominent of them is IL-8 or CXCL-8 which primarily attracts phagocytes such as
neutrophils and macrophages. Chemokines are divided into groups depending on their
structure. IL-8 belongs to the chemokine group with one amino acid residue (X) between the
first two cysteins (C) and has therefore got the new name CXCL-8. IL-8 is secreted by
leukocytic cells such as monocytes, macrophages, neutrophils, T-cells and NK-cells; and
somatic cells like endothelial cells, fibroblasts and epithelial cells [29]. Secretion of IL-8 is
activated by pro-inflammatory cytokines (IL-1, TNF-�), bacterial (LPS) or viral products via
the transcription factors NF-�B and AP-1 [29]. The main effect of IL-8 is the recruitment of
neutrophils and monocytes to the site of infection. IL-8 also plays an important role in
neutrophil activation by increasing degranulation, oxidative burst and intracellular calcium
concentrations as well as enhancing the killing of intracellular pathogens such as Candida
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albicans [29]. Basophils, eosinophils, T-cells and B-cells have also been reported to follow
IL-8 gradients [29]. IL-8 can selectively inhibit IL-4 induced IgE production, and mice
deficient in CXCR2 (the receptor for IL-8) showed increased IgE production in allergen-
induced lung inflammation [29]. In the gut, IL-8 has been shown to play an important role in
mucosal healing [30]. IL-8 enhances the migration of intestinal epithelial cells (IEC) in vitro
[31]. Furthermore, the secretion of IL-8 by IEC was shown to be sufficient to recruit
neutrophils into the subepithelial matrix, but neutrophil activation, transepithelial transversion
and resulting tissue damage were not observed [32, 33]. The role of IL-8 in tumor biology is
at least two-sided. On the one hand, IL-8 recruits neutrophils, which can directly kill tumor
cells [29]. On the other hand, the potent angiogenic activity of IL-8 may induce
neovascularization of the tumor thereby promoting tumor survival. IL-8 has also been shown
to induce the expression of matrix metallo-proteinases, which are related to metastasis [29].
Interestingly, oxygen deprivation has been reported to be an activating factor for IL-8
secretion by tumor cells [29]. Malignant transformations in some cell lines have been linked
to constitutive activation of the transcription factors NF-�B and AP-1, which amongst other
things results in increased IL-8 production [29]. Most malignant colonic epithelial cells have
been reported to overexpress IL-8 [34].
2.2.2 TNF-�
TNF-� is a pro-inflammatory cytokine with diverse biological effects, which was originally
identified as an anti-tumor agent [35]. Local production of TNF-� plays an important role in
containment and elimination of local infections [28]. However, systemically released TNF-�
is responsible for substantial pathology in connection with septic shock [36]. The expression
of TNF-� is therefore tightly regulated on the transcriptional, translational and post-
translational level [35]. Monocytes and macrophages release TNF-� in response to stimuli
such as LPS [35]. TNF-� secretion by T-cells is initiated by T-cell receptor activation. Also
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B-cells, NK cells and some non-immune cells can produce TNF-� [35]. The diverse effects of
TNF-� are mediated by two receptors TNFR1 and TNFR2, which are present on basically all
cell types [35]. The effect of TNF-� on macrophages includes the increased production of
cytokines, enhanced phagocytosis and anti-microbial response [36]. Endothelial cells
upregulate leucocyte adhesion molecules in response to TNF-� and contribute thereby to
leucocyte recruitment [36]. Furthermore, TNF-� is also involved in cell proliferation,
differentiation and death through apoptosis [36]. The anti-tumor effect of TNF-� has been
suggested to be mediated by direct cytotoxicity against tumor cells, activation of immune
responses against the tumor and damage of tumor blood vessels [35]. TNF-� is also involved
in chronic inflammatory diseases such as rheumatoid arthritis and Crohn`s disease [36].
2.2.3 IL-1�
The cytokines IL-1� and IL-1� display the same biological activities and bind to the same
receptor complex. Both cytokines are synthesized as pre-cursor proteins that have to be
cleaved into their active forms in order to be secreted [36]. However, most cells are unable to
process pro-IL-1�, which therefore only shows local activity as a membrane bound cytokine
[36]. IL-1� is one of the major cytokines released by monocytes and macrophages in response
to LPS [28]. Locally, IL-1� contributes to pathogen clearance by activation of macrophages
and T-cells. IL-1� increases the recruitment of inflammatory cells by activation of the
vascular endothelium and matrix metallo-proteinases that lead to local tissue destruction
thereby increasing the influx of effector cells [28, 36]. Systemically, IL-1� induces fever by
binding to cells of the hypothalamus and mobilizes neutrophils from the bone marrow [28].
Furthermore, IL-1� stimulates the production of IL-6, which in turn activates liver cells to
secrete acute phase proteins, thereby contributing to pathogen clearance [28].
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2.3 The gut immune system
The mucosal immune system constitutes the largest immune tissue of the body due to its
extensive exposure to antigens [28]. The gut immune system has to accomplish the task of
protecting the enormous surface area against the invasion by pathogens and maintaining
tolerance towards commensal bacteria and food antigens to avoid tissue damage. Peyer's
patches in the small intestine, solitary lymphoid follicles over the whole intestine and
mesenteric lymph nodes form the organized lymphoid tissue of the mucosal immune system
in the gut. Figure 2 illustrates the organization of a Peyer's patch. The epithelium covering
Peyer`s patches and isolated lymphoid follicels contains specialized epithelial cells called
microfold cells (M cells). Unlike normal enterocytes M cells are not covered by mucus and
lack a thick surface glycocalix [37]. M cells are specialized for the uptake and transcellular
transport of antigens and macromolecules from the gut lumen.
Figure 2: Schematic illustration of the organization of a Peyer`s patch follicle from [28]
Scattered immune cells can be found in the epithelial lining or in the lamina propria below. In
the human gut there are approximately 10-20 intraepithelial lymphocytes (IEL) per 100
enterocytes [38]. Over 80% of the IEL are CD8+ T-cells with a high proportion of innate like
T-cells [28]. Throughout the entire intestinal tract DC can protrude their dendrites through the
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epithelial lining to sample the gut lumen for antigens [38]. Intestinal macrophages are located
in the subepithelial area where they can phagocytose microorganisms that have crossed the
epithelial barrier [38]. The lamina propria also contains CD4+ T-cells, CD8+ T-cells, plasma
cells, DC, mast cells and eosinophils [28]. The organization of the mucosal immune system is
illustrated in figure 3.
Figure 3: Illustration of the organization of the mucosal immune system, modified from [28]
The mucosa contains a high number of immune cells in the absence of disease or
inflammation. This is also reflected in the high number of IgA producing B-cells in the lamina
propria, an antibody type that is non-inflammatory and can be translocated to the gut lumen
via a special secretion system of IEC [28, 38]. Maintaining intestinal immune homeostasis is
an active process that involves commensal bacteria and IEC [39-41]. IEC do not only provide
a physical barrier to the gut lumen, recent research findings suggest that they play a primary
role in initiating innate immune responses and maintaining homeostasis [39, 40]. One
important role of IEC in this respect is the “conditioning” of DCs to drive non-inflammatory
TH2 type responses [42]. This process is counteracted by basolateral exposure of IEC to
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bacteria, which restores the ability of DC to drive protective TH1 responses to bacterial
pathogens [42]. IEC express TLR-3 (recognizing double stranded RNA) and TLR-5
(recognizing bacterial flagellin) and low levels of TLR-2 and TLR-4 [40]. TLR-3 is located
intracellular at endosomal membranes, while TLR-2, 4 and 5 are expressed on the cell surface
[40]. It has been reported that the apical activation of TLR-5 on IEC lead to expression of
anti-apoptotic genes, while basolateral TLR-5 ligation induces pro-inflammatory responses
[40]. Besides compartmentalization, different responses can also be evoked by the length of
exposure. The stimulation of IEC with TLR-4 ligands e.g. induces proliferation, NF-�B
activation and inflammatory cytokine secretion while persistent exposure has an inhibitory
effect [40]. TLR signaling may also be important for epithelial cell function as TLR-2
signaling was shown to decrease permeability of the epithelial layer and maintain its integrity
[40]. It has been suggested that the composition of the diet influences IEC gene expression
and signaling to immune cells [43]. In particular the SCFA butyrate was shown to upregulate
IL-8 secretion by IEC [43]. Targeted expression of the mouse equivalent to IL-8 (MIP-2) in
epithelial cells of the small intestine and proximal colon resulted in targeted neutrophil and
lymphocyte infiltration [43]. One may therefore hypothesize, that immune-modulating dietary
components such as �-glucans could influence the complex network of IEC and underlying
immune-cells.
3. �-glucan
3.1 �-glucan structure and structure-function relationships
�eta-glucans form a very heterogeneous group of carbohydrate polymers found in the cell
walls of yeast, fungi, brown algae, and cereal grains or as extracellular polysaccharides
produced by certain bacteria. Beta-glucans from different sources share the common structure
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of �-linked glucose monomers, but differ in linkage type and side chains. Differences in �-
glucan structure for �-glucans from different sources are schematically illustrated in figure 4.
Figure 4: Schematic illustration of the �-glucan structure of �-glucans from different sources
adapted from [44].
Cereal �-glucans are linear mixed linked polymers consisting of blocks of �-(1,3)-linked
cellotriosyl and cellotetraosyl units [45]. They are soluble in hot water, have high molecular
weight (MW) in their native form in the grain cell walls and primarily exhibit random coil
conformation in solution [23, 46-48]. The properties of cereal �-glucans are discussed in more
detail in Paper I (review paper). Curdlan, a linear �-(1,3) linked polymer from Alcaligenes
faecalis, is the best known bacterial �-glucan, while the small, soluble laminarin is the most
common algae derived �-glucan. The structure of laminarin consists of a �-(1,3)-linked
backbone with a low degree of branching (DB) of single �-(1,6) linked residues [49]. Fungal
�-glucans are branched polysaccharides with a backbone of �-(1,3)-linked glucose monomers
and �-(1,6)-linked side chains of varying length and distribution [50]. Among the fungal �-
glucans, �-glucans from yeasts usually contain longer side chains than mushroom derived �-
glucans which may have single �-glucopyranosyl residues as side chains giving them a comb
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like structure [50-52]. The detailed structure of cereal and yeast �-glucans is shown in
figure 5.
Figure 5: Structural properties of cereal and yeast �-glucans adapted from [44]
Molecular weight, molecular structure, solubility and conformation in solution (triple helix,
single helix or random coil), which may actually depend on each other, and co-extracted
compounds are all supposed to be important for �-glucans biological effects. In a review from
1995 Bohn and BeMiller summarized the structure-function relationships of fungal �-glucans
with a comb like structure on anti-tumor activity and concluded that DB of 0.2-0.33, high
molecular weight and a triple helical conformation were important [51]. Also later studies
found a correlation between triple helical structure and anti-tumor activity or cytokine
secretion [52, 53]. However, the use of alkali treatment to disrupt the triple helical structures
may also have affected the activity of contaminants like LPS. This is further strengthened by
the fact that re-naturation of the triple helical structure after alkali treatment was unable to
recover the previously observed immune modulating effect [52]. Accordingly, the extent to
which the degree of branching, triple or single helix conformation and molecular weight
influence the immune modulating properties of �-glucans is still debated. The influence of
these parameters likely depends on the immune activation pathway studied, including effector
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cells, specific �-glucan receptors, way of administration (oral or parenteral), animal model
(genetic background), tumor or infection type. So far, only differences in the ability to
activate the �-glucan receptor dectin-1 for particulate versus soluble �-glucans have been
clearly demonstrated and linked to their respective mechanisms in tumor therapy [21, 54].
Table 1 gives an overview over the most commonly used �-glucan preparations in immune
modulation studies.
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Tabl
e 1:
Com
mon
ly u
sed
�-gl
ucan
pre
para
tions
in im
mun
e m
odul
atio
n. H
igh
MW
> 5
00kD
a; lo
w M
W <
100
kDa.
Ref
eren
ces g
ive
exam
ples
of s
tudi
es u
sing
the
part
icul
ar p
repa
ratio
n. Y
east
�-g
luca
n pr
epar
atio
ns c
an b
e fo
und
unde
r diff
eren
t tra
de n
ames
.
Nam
e So
urce
Sp
ecia
l str
uctu
re/p
urity
M
W
Solu
bilit
y an
d co
nfor
mat
ion
in
wat
er
Refe
renc
es
Yeas
t; �-
(1,3
)link
ed b
ackb
one
with
long
�-(1
,6)-l
inke
d sid
e ch
ain
bran
ches
Zym
osan
Sa
ccha
rom
yces
cer
evisi
ae
Crud
e ce
ll w
all p
repa
ratio
n,
cont
aini
ng m
anno
se e
tc.
Pa
rtic
ular
, uns
olub
le
[55-
59]
WGP
(who
le g
luca
n pa
rtic
le)
Sacc
haro
myc
es c
erev
isiae
Pu
re �
-glu
can
prep
arat
ion
High
Pa
rtic
ular
, uns
olub
le
[19,
21,
54]
PGG
Sa
ccha
rom
yces
cer
evisi
ae
150
kDa
Solu
ble
[21,
60]
Gl
ucan
pho
spat
e Sa
ccha
rom
yces
cer
evisi
ae
Chem
ical
ly m
odifi
ed
Solu
ble
[61,
62]
M
ushr
oom
/Fun
gus;
�-(1
,3)-l
inke
d ba
ckbo
ne w
ith sh
ort �
-(1,6
)-lin
ked
side
chai
n br
anch
es
Lent
inan
Le
ntin
us e
dode
s (Sh
iitak
e m
ushr
oom
) Si
ngle
�-(1
,6)-l
inke
d gl
ucos
e re
sidue
s
May
form
trip
le
helic
es
[63-
65]
SSG
(scl
erot
inia
sc
lero
tioru
m g
luca
n)
Scle
rotin
ia sc
lero
tioru
m
Sing
le �
-(1,6
)-lin
ked
gluc
ose
resid
ues,
DB
0.33
N
ativ
e hi
gh
Solu
ble,
form
s trip
le
helix
in so
lutio
n [6
1, 6
2, 6
6]
SPG
(Son
ifila
n/Sc
hizo
phyl
lan)
Sc
hizo
phyl
lum
com
mun
e Si
ngle
�-(1
,6)-l
inke
d gl
ucos
e re
sidue
s, D
B 0.
33
So
lubl
e, tr
iple
hel
ix in
so
lutio
n [6
7, 6
8]
Bact
eria
l; lin
ear �
-(1,3
)-lin
ked
glu
can
Curd
lan
Alca
ligen
es fa
ecal
is Hi
gh
Uns
olub
le
[59,
69-
71]
Seaw
eed;
�-(1
,3)-l
inke
d gl
ucan
with
som
e �-
(1,6
)-lin
ked
bran
ches
Lam
inar
in
Lam
inar
ia d
igita
ta
Sing
le �
-(1,6
)-lin
ked
gluc
ose
resid
ues,
DB
<0.1
5 Lo
w
Solu
ble
[54,
61,
62,
72]
Cere
als;
line
ar m
ixed
link
age
�-(1
,4)-�
-(1,3
) glu
cans
Barle
y an
d oa
t Ho
rdeu
m v
ulga
re L
./Av
ena
sativ
a
MW
in th
e pl
ant >
106
Da; i
n pr
epar
atio
ns
depe
ndin
g on
isol
atio
n pr
oced
ure
Larg
ely
solu
ble
[19,
63,
73-
78]
23
3.2 �-glucan receptors
Dectin-1
Dectin-1 is a non TLR pattern-recognition receptor with a single extracellular C-type lectin
like domain [79]. Dectin-1 is expressed on human macrophages, neutrophils and DC [80].
Recent investigation has revealed that dectin-1 activation requires the clustering of the
receptor in a synapse like fashion [54]. This can only be achieved by particulate �-glucan,
while soluble �-glucans, regardless of their molecular weight, remained unable to activate
dectin-1 [54]. Dectin-1 activation increases phagocytosis, triggers the release of reactive
oxygen species (ROS) and activates the transcription of cytokines [24, 54]. Dectin-1 is also
able to induce adaptive immunity as the activation of dectin-1 by particulate yeast �-glucan
has been shown to activate DC [21, 69]. �eta-glucan activated DC in turn preferentially
primed TH1 or TH17 differentiation of naïve CD4 T-cells and were found to activate CD8+ T-
cells to become cytotoxic effector cells [21, 69]. The effectiveness of particulate �-glucan
induced TH1 and cytotoxic T-cell responses to mediate tumor regression was further
demonstrated in mouse tumor models [21].
Complement receptor 3
Complement receptor 3 (CR3) is expressed on most myeloid cells like monocytes,
macrophages, DC, neutrophils and NK cells [24]. Beta-glucan binding to the lectin side of the
CR 3 receptor induces a primed state of the receptor that enables neutrophil or NK cell
mediated killing of iC3b opsonized target cells [72, 81]. In recent years, �-glucans (both from
yeast and barley) have been demonstrated to significantly increase the therapeutic effect of
anti-tumor monoclonal antibodies by priming the CR 3 receptor for complement mediated
killing of opsonized tumor cells [17-21].
24
Lactosylceramide
Lactosylceramide is a glycosphingolipid PRR found in lipid rafts of the plasma membranes of
many cell types [24, 79]. The receptor has been implicated in soluble yeast �-glucan induced
respiratory burst activity and NF-�B activation in human polymorphonuclear leucocytes [82].
Lactosylceramide has also been demonstrated to mediate NF-�B activation and cytokine
production in response to fungal �-glucan preparations in murine alveolar epithelial cells [83,
84]. Lactosylceramide is supposed to play a role in �-glucan induced immune-modulation
especially in non-immune cells [79].
Scavenger Receptors
By definition, scavenger receptors are all able to recognize modified low density lipoproteins.
Apart from that, their structures may vary greatly [79]. Scavenger receptors on human
monocytes have been shown to bind soluble yeast �-glucan, however, no specific receptor
was identified [85]. The scavenger receptor family member CD5 was later shown to bind
fungal cell wall components and facilitate Zymosan induced IL-8 secretion by CD5
transfected HEK293 cells [86].
3.3 Possible mechanisms behind immune modulation by dietary �-glucans
The immune-modulating effect of orally applied �-glucans has been demonstrated in several
animal models [18, 19, 21, 74, 77, 78, 87-92]. However, the mechanisms by which they exert
their effects are still poorly understood. Even though several receptors and signaling pathways
have been identified, the multiple steps from uptake in the intestine to the activation of
effector cells are still unknown. Figure 6 gives an illustration of dietary �-glucans possible
pathways from the gut to immune-modulation.
25
Figure 6: �-glucans possible steps from the intestine to immune-modulation
Dietary �-glucans are indigestible by mammalian enzymes and are supposed to reach the
large intestine more or less intact. Here they may be fermented by the indigenous microbiota,
which can lead to changes in bacterial composition and released metabolic compounds
(Figure 6, part A). Gastrointestinal microorganisms and their metabolites may affect the
immune system as immune-modulation is one of the specific health benefits that can be
mediated by probiotics, which by definition are live microorganism that survive
gastrointestinal passage and exert positive health effects on the host [10]. Most probiotic
microorganisms belong to the genera of Lactobacilli and Bifidobacterium [10]. Cereal �-
glucan is readily fermented and in vitro fermentation studies have shown increased production
of short chain fatty acids (SCFA) especially propionate but without selective stimulation of
the growth of Bifidobacteria or Lactobacilli [93, 94]. Even though �-glucans may not be
classic prebiotics (selectively promoting the growth of Bifidobacteria or Lactobacilli [95]),
they could still promote immune-modulation by the intestinal flora through e.g. changes in
26
SCFA production or microbiota composition other than Bifidobacteria or Lactobacilli.
However, the exploration of possible microbiota based mechanisms for �-glucans was not
part of this thesis and emphasis is instead laid on the direct interaction of �-glucans with
intestinal epithelial cells.
Direct interaction of �-glucans with immune competent cells (Figure 6, part B) can take place
locally in the gut by interaction with intestinal epithelial cells, a cell type that has been shown
to play a major role in orchestrating the gut immune system [39]. Also intra-epithelial
lymphocytes and DC that protrude their dendrites through the epithelial lining are in direct
contact with the gut lumen and therefore potential targets for direct interactions with luminal
�-glucans (illustrated in the right panel of figure 6) [38]. DC have been suggested to
contribute to �-glucan uptake by sampling of the gut lumen and transporting �-glucans to
mesenteric lymph nodes [96]. Recent studies have shown the potential of particulate yeast �-
glucan and curdlan to activate DC [21, 70]. However, the contribution of DC to �-glucan
uptake has still to be shown experimentally. How and to what extent �-glucans may enter the
blood stream is still debated [15]. Fluorescently marked barley and yeast �-glucans have been
detected in macrophages isolated from spleen, lymph nodes and bone marrow of mice
following oral administration [19]. The authors hypothesized that the �-glucans were taken up
and transported by gastrointestinal macrophages. However, since gastrointestinal
macrophages are located in the subepithelial area uptake over the intestinal cell layer must
have occurred. The presence of different fluorescently labeled fungal �-glucans in the plasma
of rats after oral administration has also been demonstrated [62]. On the other hand,
fluorescence labeling may change the properties of the polymers, especially if the fluorescent
molecules are bound to the main chain and not only to the reducing end [97]. This may alter
the mechanism of uptake. Labeling of the reducing end may influence the polymer structure
to a lesser degree. However, it increases the chance of dissociation of the fluorescent
27
molecule. Hence, fluorescent measurements may not reflect the distribution of the polymer
but the detached label or detached labeled fragment of the original polymer. Specific and
highly sensitive detection methods that do not require �-glucan derivatization may therefore
be helpful tools in further investigations on �-glucan uptake.
Figure 6, part C, illustrates the possible uptake mechanisms for �-glucans from the gut lumen.
Specialized epithelial cells (M-cells) in the follicle associated epithelium of Peyer`s patches
have repeatedly been suggested to mediate �-glucan uptake [44, 61, 66]. The possible role of
M-cells in �-glucan uptake is discussed in more detail in Paper I (review paper). However,
even though uptake of �-glucans via M-cells is a highly plausible mechanism, experimental
verification is still lacking. Besides M-cells, normal intestinal enterocytes may also play a role
in �-glucan uptake. Even though they are not specialized for the uptake of macromolecules
and antigens, enterocytes have been shown to transcytose nanoparticles [98] and protein
antigens [99], and they are highly abundant in the GI tract.
4. LPS
LPS is a part of the membrane of gram negative bacteria. Humans have evolved to react to
low levels of LPS to initiate innate immunity [100]. Especially monocytes and macrophages
respond to LPS by changing the expression of thousands of genes often by several orders of
magnitude [36]. The intestinal tissue, however, does not strongly respond to LPS [40]. LPS
induces the production of cytokines such as IL-1�, TNF-�, IL-12 and IL-8. The systemic
secretion of TNF-� by e.g. liver macrophages during sepsis is the main cause of fatal septic
shock [28]. LPS is also known to upregulate the expression of adhesion molecules on
endothelial cells, which increases leucocyte migration and to increase the expression of the
co-stimulatory molecules B7.1 and B7.2 on macrophages and DCs thereby helping to initiate
adaptive immune responses [28].
28
The LPS molecule consists of three different parts: the hydrophobic Lipid A, a core
oligosaccharide and the O-antigen or O-chain. The molecular structure of LPS is illustrated in
figure 7. While Lipid A anchors the molecule in the bacterial membrane; the O-antigen forms
the outer most part of LPS. The O-antigen consists of repeating sugar sequences of 3-5 sugar
moieties [101]. Differences in the composition of the O-antigen between bacterial species and
strains are responsible for their different antigenicity and determine the bacterial serotype
[36]. The core region is composed of unusual monosaccharides such as 2-keto-3-
deoxyoctonoic acid, which is characteristic for LPS and therefore sometimes used for LPS
quantification [102]. The Lipid A part contains several long chain fatty acid residues and is
highly conserved among gram negative bacteria [101]. Lipid A is responsible for the
induction of innate immunity by LPS [36, 101].
Figure 7: Illustration of the molecular structure of LPS and its localization in the cell wall of
gram negative bacteria, from [103]
29
The toll like receptor 4 (TLR-4) was first identified as the LPS signaling receptor through
genetic studies of mutant mice, which were completely unresponsive to LPS and had a single
mutation in the TLR-4 gene [104]. LPS signaling is now known to be mediated by the LPS
receptor complex consisting of LPS-binding protein (LBP), CD14, the lipid binding accessory
protein MD-2 and TLR-4. The circulating acute phase protein LBP is able to extract LPS
form the membrane of gram negative bacteria [36]. CD14 is a cell surface marker of myeloid
cells but can also be present in a soluble, secreted form [104]. LPS binding to TLR-4 results
in the initiation of two signaling pathways the MyD88 dependent and the MyD88 independent
pathway. In the MyD88 dependent pathway the MyD88 adaptor protein is directly recruited to
the cytoplasmic tail of TLR-4. A sequence of several adaptor proteins and kinases leads to the
activation of transcription factors such as NF-�B, AP1 and ELK-1 [36]. In myeloid cells an
additional MyD88 independent pathway exists, which starts from endocytosed TLR-4. Signal
transduction via the MyD88 independent pathway may lead to NF-�B activation or the
activation of interferon regulatory factor 3, a transcription factor promoting the expression of
type I interferons [28].
Due to its strong effects on innate immunity at very low doses (pg to ng level) LPS is a usual
confounder in in vitro immune-modulation assays. Several methods to assess the LPS content
of different samples or the contribution of LPS to the observed immune-modulation have
therefore been developed. Besides the above mentioned method making use of the unusual
sugar 2-keto-3-deoxyoctonoic acid [102], GC-MS detection of 3-hydroxy fatty acid from the
Lipid A part of LPS as methyl esters has also been used [105]. However, due to their high
sensitivity, assays based on the specific reaction of limulus amoebocyte lysate (LAL), an
extract from the amoebocytes of the horsehoe crab Limulus polyphemus, with LPS are
preferentially used [106]. LAL also contains a protein, called factor G, which reacts with �-
glucans and therefore has to be removed if the test is to be used for �-glucan preparations
30
[106]. Alternatively, the LPS inhibitor Polymyxin B is often used for indirect evaluation of
LPS contamination in cell culture tests [73, 106-108].
31
5. Aims of the study
The main aim of the study was to increase the current understanding of how dietary �-glucans
may modulate the human immune system by focusing on the first step from the gut lumen to
the activation of effector cells. Since cereal derived �-glucans are a part of our everyday diet
they received the main focus, but also �-glucans from yeast were investigated for comparison
purposes. The study comprises work on possible mechanisms of action and structure
functional relationships. The development of new methods to study �-glucan uptake and
carbohydrate specific effects in cell cultures were a major part of the project. Specifically the
aims were to:
� Investigate possible interactions of �-glucans with intestinal epithelial cells by
studying cytokine secretion from intestinal epithelial cell lines Caco-2 and HT-29
alone (mono-culture) or in co-culture with THP-1 derived human macrophages
� Develop a new method for the detection of low concentrations of cereal �-glucans in
cell culture supernatants and apply this method to study the transport of cereal �-
glucan across differentiated Caco-2 cell monolayers
� Investigate possible structure-function relationships of cereal �-glucans by using
different highly purified �-glucan samples (commercial and extracted from Norwegian
barley varieties) over a wide molecular weight range and comparing the effect with
yeast �-glucan preparations by studying cytokine secretion from different intestinal
epithelial cell lines.
� Develop tools to study the carbohydrate specific effects of yeast and cereal �-glucan
preparations in vitro taking potential contamination by e.g. LPS into account
32
6. List of papers and manuscripts
I Rieder, A., Samuelsen, A. B., Do cereal mixed-linked beta-glucans possess immune-modulating activities? Mol. Nutr. Food Res. 2012, 56, 536-547.
II Samuelsen, A. B., Rieder, A., Grimmer, S., Michaelsen, T. E., Knutsen, S. H.,
Immunomodulatory Activity of Dietary Fiber: Arabinoxylan and Mixed-Linked Beta-Glucan Isolated from Barley Show Modest Activities in Vitro. Int. J. Mol. Sci. 2011, 12, 570-587.
III Rieder, A., Grimmer, S., Kolset, S. O., Michaelsen, T. E., Knutsen, S. H., Cereal
beta-glucan preparations of different weight average molecular weights induce variable cytokine secretion in human intestinal epithelial cell lines. Food Chem. 2011, 128, 1037-1043.
IV Rieder, A., Knutsen, S. H., Ballance, S., Grimmer, S., Airado-Rodriguez, D.,
Cereal beta-glucan quantification with calcofluor-application to cell culture supernatants. Carbohydrate Polymers 2012, 90, 1564-1572.
V Rieder, A., Grimmer, S., Aachmann,F.L., Westereng, B., Kolset, S.O., Knusten, S.H., Generic tools to assess genuine carbohydrate specific effects on in vitro immune-modulation exemplified by �-glucans. Carbohydrate Polymers 2013, 92,2075-2083.
VI Rieder, A., Knutsen, S.H., Berget, I., Kolset, S.O., Grimmer, S., Cereal and yeast
�-glucan preparations modulate cytokine secretion by human macrophages, differentiated Caco-2 cells and Caco-2 macrophage co-cultures. Manuscript
33
7. Results
7.1 Review of cereal �-glucans immune-modulating properties (Paper I)
Beta-glucans are a heterogeneous group of glucose polymers with distinct structures
depending on their respective sources. However, this has not always been fully accounted for
in reviews on the immune-modulating properties of �-glucans. We have therefore chosen to
focus on cereal �-glucans in this review. Studies on cereal �-glucans have shown effects in
vitro on cytokine secretion, phagocytic activity and cytotoxicity of isolated immune cells/cell
lines, and activation of the complement system. However, not all conducted studies take the
potential contamination of test samples with e.g. LPS or other immunologically active
substances into account and therefore need to be interpreted with care. Cereal �-glucans have
been shown to increase antibody dependent cellular cytotoxicity in murine cancer models.
Animal studies further suggest a protective effect against infections caused by intestinal
parasites, bacteria or virus. However, uptake is still debated even though activity in animal
studies has been demonstrated for orally applied cereal �-glucan. The use of completely
different model systems in the currently available studies made it impossible to conclude on
clear structure-function relationships and more research is needed to clarify mechanisms of
action, uptake and possible activity of cereal �-glucans in humans.
7.2 Effect of high MW �-glucan fractions and arabinoxylan fractions isolated from
barley on IL-8 secretion by the intestinal epithelial cell lines Caco-2 and HT-29, NF-�B
activity in monocytic U937 cells and complement fixation (Paper II)
In this study we have investigated the potential immune-modulatory effects of fiber fractions
isolated from barley in different test systems. Two arabinoxylan and �-glucan rich fractions
with different purity were isolated from the Norwegian barley variety Tyra. The four fractions
34
showed no significant effect on IL-8 secretion and cell proliferation of the two intestinal
epithelial cell lines HT-29 and Caco-2. Furthermore, the fractions had no effect on basal or
LPS induced NF-�B activity in the stably transfected monocytic NF-�B reporter cell line
U937-3kB-LUC. Arabinoxylan and �-glucan rich fractions were also extracted from three
other barley varieties and their ability to fix complement via the classical pathway was
investigated. Some �-glucan fractions showed activity on the same level as the positive
control (a pectin fraction from Plantago major with known complement activating abilities
[109, 110]) and �-glucan fractions generally exhibited higher activity than arabinoxylan
fractions. Differences between the �-glucan fractions could not be ascribed to differences in
�(1,3)/�(1,4) linkage ratios or MW. The estimated MW of the arabinoxylan fractions was
significantly lower than for the �-glucan fractions and when both fibre types were included in
the statistical test a significant correlation between complement fixing activity and MW was
found.
7.3 Effect of cereal �-glucan fractions with different weight average MW (Mw) on the
cytokine secretion by intestinal epithelial cell lines Caco-2 and HT-29 (Paper III)
In order to investigate the possible relationship between immune-modulatory activity and
MW, cereal �-glucan preparations with weight average MW (Mw) of 40, 123, 245 and 359
kDa were tested for their ability to induce cytokine secretion in intestinal epithelial cells. The
yeast �-glucan preparation Zymosan was used as a positive control. Screening of the secretion
of 18 different cytokines (IL-1�, IL-1�, IL-2, IL-4, IL-5, IL-7, IL-8, IL-10, IL-12, IL-13, IL-
17, G-CSF, GM-CSF, MCP-1, MIP-1�, INF-�, TNF-�) in response to Zymosan and cereal �-
glucans of 40 and 359 kDa Mw revealed that HT-29 cells mainly secreted IL-8 and minor
amounts of IL-2, GM-CSF and INF-�. The secretion of IL-8 was increased by exposure of the
cells to all three �-gucans, while the other three cytokines were not influenced. Unpublished
35
results from Caco-2 cells incubated with Zymosan or control medium also showed that IL-8
was the main secreted cytokine, and the only tested cytokine whose secretion was influenced
by the presence of �-glucan (data not shown in paper III). Incubation of HT-29 cells with all
four cereal �-glucans and Zymosan resulted in significantly increased IL-8 secretion for
Zymosan and 40 kDa sample, slightly increased secretion for 359 kDa and no effect for 123
and 245 kDa samples. Caco-2 cells significantly increased IL-8 secretion in response to
Zymosan. None of the tested �-glucan preparation distinctively modulated IL-1� (Caco-2) or
TNF-� (HT-29) induced IL-8 secretion. IL-8 secretion by HT-29 cells in response to the 40
kDa cereal �-glucan was dose and time dependent and not significantly influenced by the
presence of the known LPS inhibitor Polymyxin B. Chemical characterization of the 40 kDa
sample showed glucose as the only sugar constituent and low or no protein contamination.
The increased IL-8 secretion in response to the 40 kDa sample was consequently attributed to
stimulation from the �-glucan molecules.
7.4 Development of a new method to measure low concentrations of cereal �-glucans in
cell culture supernatants and application to analysis of cereal �-glucan transport over
intact Caco-2 cell monolayers (Paper IV)
Fluorescence labeling of �-glucans to enable the study of �-glucan uptake may change the
chemical and biological properties of the polymer. Furthermore, a possible detachment of the
label during the study may confound the obtained results. We have therefore developed a
method to measure low concentrations of cereal �-glucans without the requirement of
derivatization. The method is based on the specific binding of the fluorescent dye calcofluor
to cereal �-glucans resulting in increased fluorescence intensity of the formed complex and
was optimized for high sensitivity. A concentration dependent spectroscopic response value
was calculated based on the use of the derivative signal to emphasize spectral information of
36
the calcofluor/�-glucan complex. This strategy combined with working in batch mode gave
rise to a lower detection limit of 0.045μg/mL. The method can be easily applied to measure
the transport of cereal �-glucans over differentiated Caco-2 cell monolayers. Hereby, the
transport experiment is carried out with underivatized cereal �-glucan, whose presence in the
basolateral media is detected with calcofluor after the end of the cell culture experiment.
Basolateral �-glucan concentrations above the detection limit could be measured for
approximately 50% of the Caco-2 cell monolayers after 9 h incubation with 1 mg/mL cereal
�-glucans of different Mw on the apical side. Basolateral �-glucan concentrations were
unrelated to punctures in the Caco-2 cell monolayers as no significant correlation between
transepithelial electrical resistance (TEER) and basolateral �-glucan could be found. In a
follow up experiment (unpublished data) the incubation time was increased to 22 h in order to
obtain basolateral �-glucan concentrations above the detection limit for more than 50% of the
filters. However, no increase in the number of filters with basolateral �-glucan concentrations
above the detection limit could be observed (data not shown in paper IV). Further
investigation revealed that the measured �-glucan in the basolateral compartment originated
from tiny contaminations introduced by the electrode, which was used to measure the TEER
at beginning and end of the experiment (data not shown in paper IV). In an experiment
conducted without the measurement of TEER none of the analyzed filters showed basolateral
�-glucan concentrations above the detection limit (data not shown in paper IV). In spite of the
absence of evidence for �-glucan transport, the data clearly demonstrate the high sensitivity of
the method.
37
7.5 Tools to study the carbohydrate specific effect of cereal and yeast �-glucan
preparations (Paper V)
Even if carbohydrate preparations from plant/fungal sources have been prepared to a high
degree of purity, observed immune-stimulation may be caused by minute sample
contaminations. Contamination with LPS is often ruled out by the use of Polymyxin B as a
LPS inhibitor and we have used this technique in paper 3 for testing the effect of the 40 kDa
cereal �-glucan sample. However, the use of targeted enzymatic degradation and careful
evaluation of the degradation products revealed that the effect of the 40 kDa sample on IL-8
secretion by HT-29 cells was unrelated to its �-glucan content. Instead low levels of LPS
contamination were detected by the limulus amoebocyte lysate test and the effect of the 40
kDa sample could be completely described by this contamination. Furthermore, the active
component was removed from the 40 kDa sample by preparative size exclusion
chromatography and subsequently identified as LPS like component by NMR spectroscopy.
Interestingly, the addition of Polymyxin B to the enzymatically degraded 40 kDa sample
suppressed its effect, and demonstrated clearly that Polymyxin B can not be regarded as a
general tool to suppress LPS effects in relevant polysaccharide samples. Antibodies against
TLR-2 and TLR-4 were unable to block the effect of E.coli LPS or active �-glucan samples.
Targeted enzymatic degradation of different yeast �-glucan preparations significantly
decreased the effect of one preparation (MacroGard), indicating an effect of the �-glucan
component of this preparation. Compared to other methods to study carbohydrate specific
effects, targeted enzymatic degradation has the advantage of directly relating an observed
effect to the carbohydrate of interest instead of aiming to exclude the effects of possible
contaminations.
38
7.6 Effect of cereal and yeast �-glucan preparations on THP-1 derived human
macrophages, differentiated Caco-2 cells and Caco-2 macrophage co-cultures (Paper VI)
We have previously found undifferentiated Caco-2 and HT-29 cells relatively unresponsive to
�-glucan preparations with very low levels of LPS contamination. However, it has been
indicated that Caco-2 cells only respond to apical stimuli in the presence of basolateral
leukocytes [111]. We therefore used differentiated Caco-2 cells alone (mono-culture) and in
co-culture with THP-1 derived macrophages to further investigate the possible interaction of
�-glucan preparations with intestinal epithelial cells. Caco-2 cells in mono- and co-culture
remained unresponsive towards apical addition of the cereal �-glucans tested. In contrast, two
particulate yeast �-glucans increased basolateral (mono-cultures) and apical (mono- and co-
culture) IL-8 secretion. Both preparations contain low levels of LPS. However, LPS was not
found to influence basolateral IL-8 secretion in monocultures and had a lower effect on apical
secretion, especially in co-culture, than the yeast �-glucans. Thus, the observed effects of the
two particulate yeast �-glucan preparations can not be solely explained by their LPS content,
leaving the possibility of a �-glucan related effect open. Incubation of macrophages with
different �-glucan preparations resulted in increased secretion of IL-8, TNF-� and IL-1�.
Beta-glucan preparations with higher LPS contamination generally resulted in higher cytokine
secretions. However, regression analyses of cytokine secretion data obtained with E.coli LPS
revealed that several �-glucan preparations resulted in considerably lower cytokine secretions
than expected based on their LPS content. This indicates that �-glucans may have a
dampening effect on LPS induced cytokine secretion by macrophages. However, more data
are needed to verify this hypothesis.
39
8. Discussion
8.1 Interaction with IEC
Beta-glucans are resistant to degradation by the enzymes of the mammalian digestive tract
and thus reach the large intestine intact. Enterocytes are not only the most abundant cell type
�-glucans can encounter during their passage through the GI tract, they also play an important
role in regulating intestinal immune responses [38, 39]. However, studies on the effect of �-
glucans on IEC are limited. Oral administration of oat �-glucan has been shown to increase
NF-�B activation in both leukocytes and enterocytes of the proximal small intestine but not in
the colon of NF-�B reporter mice [77]. Furthermore, oat �-glucan (Mw 60 kDa) enriched fecal
water from ileostomy patients consuming a �-glucan rich diet has been reported to increase
IL-8 secretion and ICAM-1 expression in selected intestinal epithelial cell lines [112]. The
effect was, however, only observed when the cells were co-stimulated with a mixture of pro-
inflammatory cytokines (TNF-�, IL-1�, INF-�). Due to the experimental design it was not
possible to conclude wether the observed effect was mediated directly by the �-glucan
contained in the fecal water (0.12 – 0.18 mg/mL) or indirectly by �-glucan induced changes in
the composition of the in fecal water.
In order to study the direct interaction of �-glucan with intestinal epithelial cell lines in vitro,
we developed a protocol to solubilize cereal �-glucans in cell culture media (Papers II, III, V
and VI). Commercial �-glucan samples of different Mw (40-359 kDa) and �-glucan isolated
from different barley varieties (Mw about 600-900 kDa) were tested. The cereal �-glucan
sample with the lowest Mw (40 kDa) significantly increased IL-8 secretion by HT-29 cells
(Paper III). However, the activity was subsequently attributed to contamination of the sample
with LPS (Paper V). None of the other tested cereal �-glucan samples significantly increased
the cytokine secretion by HT-29 or Caco-2 cells (Paper II, III and V). Stimulation of HT-29
cells with TNF-� and Caco-2 cells with IL-1� did not show any effect of the tested �-glucan
40
samples on cytokine induced IL-8 secretion (Paper III). Furthermore, apical addition of cereal
�-glucans to differentiated Caco-2 cells in mono- or co-culture with THP-1 derived
macrophages did not show any effect on cytokine secretion (Paper VI). However, our
experiments with yeast �-glucans revealed a potential of the particulate yeast �-glucan
preparations MacroGard and Zymosan to stimulate cytokine secretion by IEC. Both samples
increased IL-8 secretion by HT-29 cells, and targeted enzymatic degradation revealed that at
least the observed activity of MacroGard could be related to the �-glucan part of the sample
(Paper V). Apical addition of MacroGard or Zymosan to differentiated Caco-2 cells in mono-
culture or in co-culture with THP-1 derived macrophages resulted in increased basolateral
(mono-culture) and apical (mono- and co-culture) IL-8 secretion (Paper VI). Interestingly,
differentiated Caco-2 cells, especially when in co-culture, did not react strongly to the
presence of LPS. Thus, even though both �-glucan samples contained low levels of LPS, their
observed effects on IL-8 secretion could not be solely explained by their LPS content, leaving
the possibility of a �-glucan related effect open. On the other hand, a highly purified soluble
yeast �-glucan (Wellmune) did not elicit any effect on cytokine secretion by HT-29 cells or
differentiated Caco-2 cells in mono- or co-cultures (Paper V and VI).
Our experiments with cereal �-glucans as well as other reported studies [77, 112] suggest that
cereal �-glucans are unable to directly activate enterocytes to secret cytokines. However, even
though we did not find any effect of cereal �-glucans on IL-8 secretion by IEC lines, cereal �-
glucans may have a direct effect on other aspects of enterocyte biology. It has for instance
been shown that the consumption of oat �-glucan decreased the levels of antimicrobial
peptides in the fecal water of ileostomy patients [113]. Cereal �-glucans may activate
intestinal leukocytes, which then in turn may activate enterocytes as suggested by Volman et
al., 2010 [77]. More research is needed to increase the current understanding of the interaction
of cereal �-glucans with IEC. Our results with yeast �-glucans on the other hand, suggest that
41
particulate but not soluble yeast �-glucan preparations may increase IL-8 secretion by IEC.
Increased IL-8 secretion by IEC may be beneficial as IL-8 has been shown to play an
important role in mucosal healing [30, 31]. Furthermore, IL-8 secretion by IEC has been
shown to lead to the recruitment of neutrophils and lymphocytes [32]. One may speculate that
the increased number of immune cells in the epithelial lining may contribute to the potential
benefit of �-glucans on resistance to infection. Interestingly, oral administration of yeast �-
glucan to mice has also been reported to increase the amount of intraepithelial lymphocytes
[90].
8.2 Uptake
Uptake of �-glucans from the gut is an important aspect in the understanding of the potential
immune-modulating properties of dietary �-glucans. However, only limited information on �-
glucan uptake is available. Uptake has been indicated by the detection of fluorescein
dichlorotriazine labeled yeast �-glucan particles (WGP) and barley �-glucan in the bone
marrow, peritoneal lymph nodes and spleen of mice following oral administration [19].
However, the label fluorescein dichlorotriazine is able to covalently react with hydroxyl
groups of the �-glucan chains and may due to it`s two reactive groups also lead to cross-
linking of the polymers. The resulting �-glucan complexes may have dramatically changed
properties compared to the parent molecules, including a possible altered uptake mechanism
or receptor affinity. Interestingly, the fluorescein labeled �-glucans were also detected in
intracellular compartments in macrophages [19]. In another study Alexa Fluor 488 labeled
laminarin, scleroglucan and glucan phosphate were detected in the serum of rats after oral
administration of 1 mg/kg [61]. Labeling of only the reducing end ensured identical properties
of labeled and unlabeled polymers. Maximum detected serum concentrations of the different
compounds occurred between 3 and 4h after administration and were approximately 40, 115
42
and 355 ng/mL for glucan phosphate, laminarin and scleroglucan, respectively. The
bioavailability ranged from 0.5 to 4.9%. Alexa Fluor 488 labeled particulate yeast �-glucan
were included in the same study but could not be detected in the serum of rats. Thus, the
authors hypothesized that particulate �-glucans may be taken up and transported by
macrophages as described by Hong et al. [19]. Another group has used the reactivity of the
limulus amoebocyte lysate (LAL) factor G with �-glucan for the detection of unlabeled
soluble yeast �-glucan in rat serum after oral administration of 20 mg/kg daily over 14 days
[96]. Even though �-glucan concentrations were reported to be significantly different from the
control group, levels of only 5 ng/mL were reached. Together these studies indicate that only
low amounts of �-glucans may be taken up from the gut.
The uptake mechanisms for �-glucans are still unclear, but Rice et al. have found that glucan
phosphate is internalized by a subset of IEC through a dectin-1 independent mechanism [61].
It has been repeatedly suggested that �-glucans are taken up by M cells [44, 61, 89]. M cells
are epithelial cells specialized for the uptake and transport of macromolecules and antigens
over follicle associated epithelium throughout the intestine [37, 114]. However, even though
this is a plausible mechanism, it has never been shown experimentally. Normal IEC are highly
abundant in the gut and have been shown to transcytose soluble protein antigens and
nanoparticles [98, 99], which makes them an interesting candidate for �-glucan uptake. In
order to study the uptake of cereal �-glucans across differentiated Caco-2 cell monolayers, we
developed a new method to detect low concentrations of cereal �-glucans in cell culture
supernatants (Paper IV). The new detection method is based on the specific interaction of the
fluorescent dye calcofluor with cereal �-glucans, and the interaction is carried out after the
cellular studies. Thus, the test does not require �-glucan derivatization and consequently
avoids structural changes that may affect cellular uptake and transport. Detection of low
levels of cereal �-glucan in the basolateral compartments in the first experiments were
43
subsequently shown to be related to contamination by the electrode used for TEER
measurement, but demonstrated the applicability of the developed method. Later results
indicated the absence of cereal �-glucan transport over differentiated Caco-2 cell monolayers
or transport below the detection limit of the assay of 45 ng/mL. As outlined above the
published data on �-glucan serum concentrations after oral administration to rats vary
considerably with the �-glucan type (laminarin, scleroglucan, yeast �-glucan, glucan
phosphate) and detection method (fluorescence vs LAL test) from 5 to 355 ng/mL. It is
therefore difficult to decide if the detection limit of 45 ng/mL is too high to measure cereal �-
glucan transport across the cell layer or if the results simply indicate the absence of transport.
In the last experiment, a total of 1.5 mg cereal �-glucan added to the apical side resulted in
less than 60 ng �-glucan on the basolateral side (the addition of calcofluor and buffer solution
to the cell culture supernatant during the �-glucan assay introduced a 3:4 dilution
corresponding to an increase of the detection limit from 45 ng/mL to 60 ng/mL). This
corresponds to an uptake rate of under 0.004% in 22 h. Thus, one might speculate that the
uptake rate is too low to give physiologically relevant concentrations, and furthermore that
IEC do not contribute to a major extent to the uptake of cereal �-glucans from the gut.
Caco-2 cells can be differentiated into cells with phenotypes resembling those of small
intestinal enterocytes. The development of microvilli during the differentiation process can be
shown by scanning electron microscopy (Figure 8).
44
Figure 8: Scanning electron micrograph of differentiated Caco-2 cells showing the
development of microvilli (Rieder et al., unpublished results).
Even though differentiated Caco-2 cells are a commonly used in vitro model system for the
study of drug absorption [115-117], the reported TEER values for differentiated Caco-2 cell
layers (average 300-600 �cm2) are considerably higher than for the human intestine (average
30 �cm2) [118]. Consequently Caco-2 cell monolayers have a lower permeability towards
substances transported primarily by the paracellular route [115] and may therefore not be the
ideal model system for transport studies with hydrophilic molecules such as �-glucans, even
though paracellular transport of �-glucans might be limited by their size.
It has been shown that the presence of macrophages on the basolateral side increased the
uptake of microparticles by Caco-2 cell monolayers [119]. Furthermore, the addition of
supernatants from PBMC stimulated with wheat germ agglutinin to the basolateral side of
differentiated Caco-2 cells has been shown to alter the integrity of the Caco-2 cell monolayers
[120]. It may therefore be worthwhile to investigate the uptake of �-glucan over Caco-2 cell
45
monolayers in the presence of leukocytes. Furthermore, the use of an in vitro M cell model,
which has been established by the co-culture of differentiated Caco-2 cells with Raji B-cells
[121, 122], to study �-glucan uptake may also prove to be a useful experimental system. The
new calcofluor based method for cereal �-glucan detection may also be a helpful tool in this
undertaking.
Attempts to apply the calcofluor method for the detection of cereal �-glucan in blood have so
far been hindered by fluorescence interference of serum. However, the use of appropriate
sample preparation techniques in order to remove serum fluorescence may render the
calcofluor method useful for quantification of cereal �-glucan also in serum samples.
Compared to the LAL test for �-glucans, the calcofluor method has a higher detection limit
and can only be applied for cereal �-glucans. On the other hand, the main advantage of the
calcofluor method is the specificity of the changes induced in the emission spectra of
calcofluor by cereal �-glucan binding. Activation of the limulus coagulation factor G depends
on �-glucan structure (lower activity for cereal �-glucans than yeast �-glucan and curdlan),
molecular weight and conformation in solution [123]. Higher MW and single helix
conformation of �-glucans in solution are associated with higher reactivity with LAL [124].
Due to this dependency of the LAL test specific standard curves for each tested �-glucan are
required. Furthermore, MW and conformation in solution of the tested �-glucans have to
remain unchanged under study conditions in order to obtain exact concentrations with the
LAL test.
8.3 Macrophages
Macrophages play an important role in tissue homeostasis by clearance of apoptotic cells,
tissue remodeling and repair [125]. Activation of macrophages during infection/inflammation
plays a crucial role for pathogen clearance both by the innate and adaptive immune systems
46
[28]. Therefore the ability of �-glucans to activate macrophages has been preferentially
studied. Reports on the activation of macrophages by �-glucans include the activation of
phagocytosis [78, 126], increased secretion of cytokines [73, 126-128] and increased
production of ROS [54, 129]. The activation of cytokine secretion and ROS production have
been found to be dependent on the macrophage origin and micro-environment as well as on
the nature of the �-glucan (particulate vs. soluble) [24, 54, 126, 128]. However, even though
dectin-1 mediated cytokine secretion by macrophages has been shown to require crosslinking
of the receptor by particulate �-glucan [54], soluble oat �-glucan has been reported to increase
the secretion of IL-1 by murine peritoneal macrophages and the murine macrophage cell line
P338D1 [73]. This indicates that other mechanisms for cytokine secretion by �-glucans
besides dectin-1 signaling may exist.
We have studied the secretion of the cytokines IL-8, TNF-� and IL-1� by THP-1 derived
macrophages in response to different cereal and yeast �-glucan preparations (Paper VI). The
�-glucan samples that significantly increased cytokine secretion also contained the highest
levels of LPS (3.5-7 ng/mL). The high cytokine secretion levels induced by the LPS
containing samples also showed a relatively high variability, which may be the reason why
none of the �-glucan samples with minimal LPS contamination (0.01 to 0.04 ng/mL) had a
statistically significant effect on the cytokine secretion (Paper VI). The effect of oat and
barley �-glucan on cytokine gene expression by THP-1 derived macrophages has recently
been demonstrated with samples containing less than 1 pg LPS/mL [63]. Both extracted and
commercial cereal �-glucan samples increased the expression of the cytokines IL-1� and IL-8
and the expression of transcription factor NF-�B after 3-6 h of incubation, while the
expression of IL-10 was only upregulated after 24 h of incubation [63]. Statistical significance
testing of the observed increases compared to the control was unfortunately not performed in
this study. However, oat and barley �-glucans were shown to significantly decrease the LPS
47
induced IL-1� expression by THP-1 derived macrophages [63]. LPS induced IL-8 expression
was also reduced, however this effect was not significant [63]. In order to investigate if the
increased cytokine secretion in response to the �-glucan preparations in our own study (Paper
VI) was due to contamination with LPS, results obtained with E.coli derived LPS were used
to build models for the prediction of the LPS-induced secretion of IL-8, TNF-� and IL-1�.
These regression analyses revealed that several �-glucan preparations gave a considerably
lower cytokine secretion than expected based on their LPS content. This indicates that �-
glucan may have a dampening effect on LPS-induced cytokine secretion from macrophages,
which is in accordance with the data obtained by Chanput et al. [63]. Furthermore, two studies
have suggested that �-glucans may protect from shock and organ injury during sepsis [96,
130]. Both studies used yeast �-glucan preparations and showed an attenuated cytokine
release in the �-glucan treated group of mice, which may have contributed to the observed
protection from lung, renal and hepatic injury [96, 130]. Also oral administration of yeast �-
glucan has been shown to attenuate the increase of plasma IL-6 and TNF-� in pigs challenged
with parenteral LPS [131]. However, more research is needed to understand the effect of
different �-glucans on the cytokine secretion from macrophages in the presence or absence of
inflammation.
The majority of our work with �-glucans and cell cultures has been conducted with different
intestinal epithelial cell lines and cereal �-glucan preparations. Hence, the applied �-glucan
dose of 1 mg/mL has been chosen based on the recommended intake for cereal �-glucan of 3
g/day and 0.75 g/serving [14]. A cereal �-glucan intake of 0.75 g/serving will with an
estimated stomach volume of 750 mL result in a cereal �-glucan concentration of 1 mg/mL in
the intestine. This concentration is therefore physiologically relevant for the interaction of
cereal �-glucan with IEC. Macrophages, however, are located in the subepithelial space and
will only be exposed to the �-glucan fraction that might be taken up across the epithelial
48
layer. As outlined above, the uptake of �-glucans from the gut is still debated. However, the
few available studies on �-glucan uptake all show a very low bioavailability of �-glucans.
Hence, the concentration of 1 mg/mL which we have used for the experiments with
macrophages is probably too high in a physiological context. A dramatic reduction in �-
glucan dosage down to concentrations around 10 μg/mL would better reflect the probable in
vivo situation.
8.4 Carbohydrate specific effects
The assessment of immune-modulating properties of food or medicinal plant components
requires extremely pure preparations as co-extracted compounds or contaminants may have a
profound effect on the tested parameters, especially in in vitro test systems. One of the most
dreaded contaminants of test samples for biological activity testing is LPS, a membrane
molecule of gram negative bacteria. LPS binding to TLR-4 initiates a range of signaling
events that can lead to the activation of several transcription factors such as NF-�B and AP-1,
which may further lead to the secretion of pro-inflammatory cytokines [36]. In addition, LPS
is known to increase phagocytosis and nitric oxide production by e.g macrophages [28, 132].
Thus, the cellular responses towards LPS and immune-modulating food/plant components
may be difficult to distinguish from each other at first glance.
The addition of the antibiotic Polymyxin B to cell cultures is commonly used to eliminate the
effect of possible LPS contaminations [73, 106-108]. Polymyxin B binds to the Lipid A part
of the LPS molecule [133] thereby inhibiting its biological activity [134]. However, we have
shown that Polymyxin B was unable to suppress the activity of LPS found in a cereal �-
glucan preparation (Paper V). The activity of LPS in this sample could, however, be
suppressed by the addition of Polymyxin B after extensive �-glucan depolymerization into
oligosaccharides with lichenase. This indicates that polymeric �-glucan samples may complex
49
LPS and make it unavailable for binding by Polymyxin B (Paper V). Therefore, an important
conclusion from our studies is that the sole use of Polymyxin B is not sufficient to rule out
LPS contamination of polymeric samples.
In Paper V we showed that targeted enzymatic degradation can be a good strategy to assess
the carbohydrate specific effect of polymeric samples. This approach has the main advantage
of attempting to detect a direct effect of the polymer of interest and can therefore both address
the possible effect of a potential LPS contamination and also exclude the potential
contribution of other sample components like for instance co-extracted compounds (Paper V).
The application of this approach to samples intended for cell culture testing, however, is not
without challenge as some enzyme preparations have been shown to influence cell viability
(Paper V). Membrane filtration of the enzyme preparations that removed their low molecular
compounds with negative effects on the cells but retained enzymatic activity was successfully
applied to overcome this challenge (Paper V).
In a different approach we tested the ability of a TLR-4 blocking antibody to suppress LPS
activity, although without success (Paper V). Even though the same antibody clone has been
previously described to reduce the effect of LPS on cytokine secretion by various cell types
[135-137], we did not observe any effect on LPS-induced IL-8 secretion by HT-29 cells
(Paper V, unpublished data). HT-29 cells have been reported to contain considerable amounts
of intracellular TLR-4 protein while cell surface expression is low [138], which may explain
the failure of the TLR-4 antibody to block LPS-induced cytokine secretion from HT-29 cells.
However, none of the other three studies that showed a suppression of LPS activity following
addition of the TLR-4 antibody could report a complete abrogation of the LPS effect [135-
137]. The combined use of different TLR-4 antibodies, binding to different epitopes of the
receptor, may increase the blocking potential. Alternatively, the use of specific cell lines that
do not express TLR-4 may be a possibility.
50
In a third approach, we used E.coli derived LPS in concentrations corresponding to the range
of the contamination levels of the �-glucan samples to compare LPS and �-glucan effects
(Paper V and VI). Simple comparison of the effects obtained with LPS and �-glucan can give
a good first indication if observed effects may be due to LPS contamination in the sample. In
Paper VI we used regression analysis to further evaluate if the observed increase in cytokine
secretion by macrophages in response to �-glucan preparations was due to their LPS content.
We found that several �-glucan preparations gave a considerably lower cytokine secretion
than expected based on their LPS content. This indicates a potential dampening effect of �-
glucan on LPS induced cytokine secretion from macrophages. Even though more data is
needed to verify this hypothesis, regression analysis of cytokine secretion evoked by LPS
standards may become an interesting tool to investigate the effect of test samples in spite of
LPS contamination.
Our results clearly show that LPS contamination of �-glucan samples is a real problem for in
vitro test systems and has to be carefully evaluated in order to exclude false positive results.
However, we did find that the effect of particulate yeast �-glucan (MacroGard) on cytokine
secretion by different IEC systems was related to the �-glucan component of this preparation.
Others have also reported immune-modulating properties of �-glucan preparations with very
low LPS contamination (10 pg/mg or lower) [54, 63, 73, 78, 91, 92]. It is therefore unlikely
that the immune-modulating properties of �-glucan preparations shown in animal studies are
only caused by LPS contaminations.
A huge array of different LPS detection methods have been developed [102, 105, 139-142].
However, assays based on the ability of LPS to activate clotting enzymes of the haemolymph
of the horseshoe crab Limulus amoebocyte [141, 143] are preferentially used due to their high
sensitivity and easy application as several commercial assay kits based on the Limulus
amoebocyte lysate (LAL) are available. LAL also contains a factor, factor G, that reacts with
51
�-glucans (from yeasts, mushrooms and cereals) in a clotting reaction similar to the one
induced by LPS [123, 124]. Commercial test kits where factor G has been removed are
available and can be used to quantify LPS in �-glucan samples. However, the solubility of �-
glucan preparations is sometimes limited and aggregate formation between polymeric �-
glucan and LPS may occur. To what extent this may influence the LAL test can only be
speculated upon. In our experiments we could detect considerable amounts of LPS (7 ng/mg)
in the 40 kDa cereal �-glucan samples, even though Polymyxin B was unable to suppress the
effect of 40 kDa on cytokine secretion (Paper VI). One may therefore speculate that complex
formation between �-glucan and LPS has less influence on the LAL test than on Polymyxin B
binding. Repeated use of the LAL test kit and a critical evaluation of the results revealed a
high overall standard deviation of 36%, which may be partly related to solubility/availability
problems of LPS in polymeric �-glucan samples or due to the fact that small amounts of
factor G, which reacts with �-glucan were not completely removed by the manufacturer.
However, the fact that some of the analyzed �-glucan samples (Wellmune and 123 kDa)
showed very low LPS contents (0.01 ng/mg) makes it unlikely that traces of factor G in the
assay may have caused considerable false positive results in the other �-glucan preparations.
An interesting hypothesis that exposure to LPS can have beneficial effects for human health
has recently been put forward [103, 144, 145]. LPS is supposed to play a role in homeostasis
of the immune system of the intestine [145]. Furthermore, Inagawa et al. have summarized
several animal studies that show a beneficial effect of oral LPS administration on various
infectious diseases [103]. Oral administration of 20 ng/mL LPS in the drinking water, for
example, was shown to protect mice from Toxoplasma gondii infection [146]. Interestingly,
LPS exposure or oral administration has also been shown to have some anti-carcinogenic
potential [144]. On the basis of this potential health effect of LPS it may be speculated that �-
glucan preparations act as LPS carriers in vivo and thereby mediated some of their reported
52
beneficial effects. Furthermore, the LPS doses that can be achieved by LPS contaminated �-
glucan preparations (based on our results for LPS content in different �-glucan preparations)
are in the same range as the LPS doses (20 ng/mL) that have been reported to be effective in
animal studies [146].
8.5 Beta-glucans structure-function relationship
The anti-tumor activity of fungal �-glucans has been shown to depend on their DB, MW and
helical conformation [51]. Furthermore, fungal �-glucans with a higher DB, higher MW and a
single rather than triple helical conformation in solution have been shown to be more potent
activators of LAL [124]. Cereal �-glucans behave mostly like random coil polysaccharides in
solution [48]. However, they can vary considerably in MW and fine structure. The ratio of �-
(1,3)-linked cellotriosyl to cellotetraosyl units for example varies for �-glucans from different
cereals [46]. The cereal �-glucan preparations we used did not only vary in Mw but also
showed differences in fine structure as revealed by the different ratio of oligosaccharides with
degree of polymerization (DP) of 3 and 4 released by lichenase (Paper V and Table 2).
Table 2: Molar ratio of oligosaccharides released by lichenase treatment of different �-glucan preparations as determined by high performance anion exchange chromatography with pulsed amperometic detection (described in Paper V).
�eta-glucan standard DP3/DP4 molar ratio 40 kDa 1.78 123 kDa 1.92 245 kDa 2.80 359 kDa 3.19
As described before, none of the tested cereal �-glucan preparations, regardless of their Mw or
fine structure, showed any effect on cytokine secretion in the different IEC line test systems
that could be related to the �-glucan component of the sample (Paper III; V and VI).
Furthermore, �-glucan fractions isolated from barley showed no effect on basal or LPS
induced NF-�B activation in monocytic U937 cells stably transfected with a NF-�B reporter
53
gene (Paper II). Nevertheless, two of the tested cereal �-glucan preparations, 359 kDa and
BG_STS (extracted from barley with an approximate Mw of 600 kDa), showed a potential to
dampen LPS induced cytokine secretion by macrophages (Paper VI). However, more data is
needed to verify this hypothesis and investigate a possible structure-function relationship of
cereal �-glucans in this respect. Interestingly, �-glucan fractions extracted from different
barley varieties were found to activate complement (Paper II), but differences in complement
fixing ability could not be ascribed to MW or fine structure (Paper II).
Interestingly, two particulate yeast �-glucans (MacroGard and Zymosan) significantly
increased IL-8 secretion by HT-29 and Caco-2 cells in mono- and co-culture, while a soluble
yeast �-glucan preparation (Wellmune) elicited no effect in the same test systems (Paper V
and VI). Since cereal �-glucans are largely soluble in water and have been used in a soluble
form in this study, it might be speculated that the physical properties (soluble vs. particulate)
more than the chemical structure (DB, MW, type of linkages) of different �-glucans
determine their specific activity. The recent finding that the known �-glucan receptor dectin-1
can only be activated by particulate �-glucans may strengthen this hypothesis [54]. However,
structure-function relationships of �-glucans are likely to depend on the studied immune
activation pathway, cellular or animal model system, involved �-glucan receptors, and way of
administration (oral or parenteral). Thus the complex relationships between �-glucans
chemical and physical properties and their immune-modulating potential are still far from
understood. A more general discussion of cereal �-glucans structure-function relationship in
immune modulation involving more results from other studies can be found in Paper I.
8.6 Current knowledge of dietary �-glucans mechanisms of action
Cereal as well as baker`s yeast (Saccharomyces cerevisiae) �-glucans are a part of the human
diet. Both types of �-glucans have been shown to be effective against different types of
54
infection [74, 91, 92, 147-151] and they have been shown to enhance the activity of anti-
tumor antibodies in different animal models [17-21, 152]. The mechanisms of action behind
this synergistic effect in animal models of tumor therapy have been partially elucidated. Beta-
glucan binding to the lectin-side of CR3 on neutrophils and NK cells induces a primed state of
the receptor, which enhances the killing of iC3b opsonized target cells [19, 21, 81]. This
increases antibody dependent cellular cytotoxicity, which in combination with the activation
of complement deposition on tumor cells initiated by anti-tumor antibodies facilitates tumor
killing [19, 21].
It is possible that the anti-infective properties of cereal and yeast �-glucans partially rely on a
similar mechanism, which increases killing of complement coated pathogens. However, this
has never been shown experimentally. Apart from a potential microbiota based mechanism,
which has not been addressed in this thesis, dietary �-glucans can initiate immune-modulation
either by interacting with specific cells of the GI tract or by uptake across the intestinal
epithelium. We have demonstrated the potential of particulate yeast �-glucans to increase IL-8
secretion by IEC lines, which may also be an important mechanism for dietary yeast �-
glucans immune-modulating effect. On the other hand, our data do not indicate any effect of
cereal �-glucans on cytokine secretion by IEC nor uptake of cereal �-glucans across IEC
(Paper II, III, V, VI and additional data presented together with Paper IV). However,
interaction of dietary cereal �-glucans with IEL or uptake over the intestinal epithelium via M
cells and subsequent activation of macrophages could be other possible explanations for the
protective effect of cereal �-glucans demonstrated in animal models and should be further
investigated. Another possibility is the activation of DC that protrude their dendrites through
the epithelial lining in the gut by cereal or yeast �-glucans, as both types of �-glucans have
been shown to activate DC to express the co-stimulatory molecule CD86 in vitro [15].
55
We also found indications for a dampening effect of some cereal and yeast �-glucan
preparations on LPS induced cytokine secretion by macrophages (Paper VI). Increased
phagocytic activity and cytokine secretion by macrophages in response to cereal and yeast �-
glucans have been repeatedly demonstrated in vitro [63, 73, 78, 153]. It seems therefore, that
macrophages may play an important role in mediating �-glucans immune-modulating effects.
However, more research is needed to understand the protective effects that cereal and yeast �-
glucans have shown against various diseases in animal models. A more detailed knowledge of
the potential mechanism behind the activity of these two types of �-glucans deducted from
animal and in vitro models will undoubtedly be helpful for the subsequent conduction of
meaningful human studies. So far, dietary yeast �-glucans have been reported to increase
salivary IgA concentrations in healthy volunteers and protected against exercise induced
depletion of monocytes in healthy subjects [154, 155]. Oral application of cereal �-glucan on
the other hand did not show any effect on C-reactive protein level or LPS induced cytokine
secretion by PBMC in hyper-cholesterolemic subjects or exercise induced immune-changes of
trained cyclists [156-158].
56
9. Concluding Remarks
We have shown that cereal �-glucans of different origin, fine structure and molecular weight
are unable to modulate cytokine secretion by several IEC line systems including a co-culture
system of differentiated Caco-2 cells with THP-1 derived human macrophages. Furthermore,
the absence of cereal �-glucan uptake across differentiated Caco-2 cell monolayers indicated
that IEC may not play a major role in cereal �-glucan uptake from the intestine in vivo. The
developed method for cereal �-glucan quantification in cell cultures, based on the interaction
of cereal �-glucans with the fluorescent dye calcofluor, may be a useful tool to study cereal �-
glucan uptake in other cellular systems e.g. in vitro M cell models. Despite the
unresponsiveness of IEC line systems towards cereal �-glucans, these polysaccharides were
found to activate complement via the classical pathway and showed a potential to reduce LPS
induced cytokine secretion by THP-1 derived macrophages.
We have shown that particulate, but not soluble yeast �-glucans increase IL-8 secretion by
IEC lines, including basolateral secretion of IL-8 by differentiated Caco-2 cell mono-cultures.
Increased IL-8 secretion by IEC in vivo may be an important mechanism for the immune-
modulating effect of dietary yeast �-glucans.
We clearly showed the necessity to validate observed immune-modulating properties of
carbohydrate preparations from plants/fungi by investigating their carbohydrate specific
effects. To avoid false positive results a relationship between the carbohydrate
polymer/structure and the observed effect has to be established. We have demonstrated that
the use of Polymyxin B is insufficient to rule out LPS contamination of polymeric samples
like �-glucans. Instead targeted enzymatic degradation of the component of interest is
recommended since this method has been proven to be a powerful tool to assess possible
57
carbohydrate specific effects and hence to validate results obtained with polysaccharide
preparations.
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10. Reference List
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Int. J. Mol. Sci. 2011, 12, 570-587; doi:10.3390/ijms12010570�
International Journal of
Molecular Sciences ISSN 1422-0067
www.mdpi.org/ijms Article
Immunomodulatory Activity of Dietary Fiber: Arabinoxylan and Mixed-Linked Beta-Glucan Isolated from Barley Show Modest Activities in Vitro
Anne Berit Samuelsen 1,*, Anne Rieder 2, Stine Grimmer 2, Terje E. Michaelsen 1,3 and Svein H. Knutsen 2
1 Pharmacognosy, Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, Oslo N-0316, Norway
2 Nofima Mat, Norwegian Institute of Food, Fisheries and Aquaculture Research, Aas N-1430, Norway; E-Mails: [email protected] (A.R.); [email protected] (S.G.); [email protected] (S.H.K.)
3 Norwegian Institute of Public Health, Oslo N-0403, Norway; E-Mail: [email protected]
* Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +47-22-856-568; Fax: +47-22-854-402.
Received: 26 November 2010; in revised form: 20 December 2010 / Accepted: 4 January 2011 / Published: 18 January 2011
Abstract: High intake of dietary fiber is claimed to protect against development of colorectal cancer. Barley is a rich source of dietary fiber, and possible immunomodulatory effects of barley polysaccharides might explain a potential protective effect. Dietary fiber was isolated by extraction and enzyme treatment. A mixed-linked �-glucan (WSM-TPX, 96.5% �-glucan, Mw 886 kDa), an arabinoxylan (WUM-BS-LA, 96.4% arabinoxylan, Mw 156 kDa), a mixed-linked �-glucan rich fraction containing 10% arabinoxylan (WSM-TP) and an arabinoxylan rich fraction containing 30% mixed-linked �-glucan (WUM-BS) showed no significant effect on IL-8 secretion and proliferation of two intestinal epithelial cell lines, Caco-2 and HT-29, and had no significant effect on the NF-�B activity in the monocytic cell line U937-3�B-LUC. Further enriched arabinoxylan fractions (WUM-BS-LA) from different barley varieties (Tyra, NK96300, SB94897 and CDCGainer) were less active than the mixed-linked �-glucan rich fractions (WSM-TP and WSM-TPX) in the complement-fixing test. The mixed-linked �-glucan rich fraction from NK96300 and CDCGainer showed similar activities as the positive control while mixed-linked �-glucan rich fractions from Tyra and SB94897 were less active. From these
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results it is concluded that the isolated high molecular weight mixed-linked �-glucans and arabinoxylans from barley show low immunological responses in selected in vitro test systems and thus possible anti-colon cancer effects of barley dietary fiber cannot be explained by our observations.
Keywords: arabinoxylan; mixed-linked �-glucan; barley; Caco-2; complement-fixing test; dietary fiber; HT-29; IL-8; U937; NF-kappaB
1. Introduction
Dietary fiber have been claimed to protect against the development of colorectal cancer (CRC) [1], but according to several reviews, evidence of such a relationship is scarce [2–4]. CRC is one of the most common types of cancer world-wide, and also in Norway, the incidence of CRC has increased over the past 50 years. The reason for this is largely unknown, but lifestyle and diet probably contribute [5,6].
Chronic inflammation is associated with increased risk of cancer development [7], and patients with inflammatory bowel diseases, such as ulcerative colitis and Crohn’s disease, have increased risk of developing CRCs [8]. Plasma levels of the acute phase protein C-Reactive Protein (CRP) which is a marker of inflammation, are elevated in persons who subsequently develop CRC [9]. Increased intake of dietary fiber reduces CRP levels [10–12] as well as the levels of the proinflammatory cytokines IL-6 and TNF� [13]. Strengthening the immune system’s ability to detect and eliminate cancer cells, a process called cancer immunosurveillance [14], on the other hand may have a protective effect. The potential of dietary fiber to promote cancer immunosurveillance is currently unknown. However barley beta-glucan has been shown to increase the effect of anti-tumor antibodies in mice [15,16]. In general, dietary fiber may affect inflammatory processes and immune responses by several mechanisms. Amongst the most studied are the mechanisms exerted by butyrate, a short chain fatty acid produced in the colon following fermentation of dietary fiber. Butyrate has anti-inflammatory [17], apoptotic, and anti-proliferative activities on cancer cells [18,19]. Dietary fiber, depending on their structures, can affect the intestinal immune system by being taken up by M-cells in the Peyer’s patches and transported to underlying immune cells and other cells. This may result in a local cytokine production which can influence T-cells, B-cells, antigen presenting cells and other immune cells. Fiber may also be taken up by intestinal macrophages or dendritic cells (i.e., antigen presenting cells) and transported to lymph nodes, spleen and bone marrow [20,21]. In addition, direct interaction of fiber with colonic epithelial cells or leukocytes may induce changes in immune reactions relevant for inflammation and the development of cancer.
Barley (Hordeum vulgare) is an interesting source of dietary fiber and was previously the preferred grain for food in the Nordic region; mainly due to its short growing season due to the climate. In Norway, barley is still the major cereal crop, but only a part is used for human consumption, the majority is used as animal feed. Barley as well as oats are rich in dietary fiber, mainly mixed-linked �-glucans and arabinoxylans [22,23]. In these cereals, �-glucans are linear �-(1�3)/(1�4)-D-glucopyranosyl polymers referred to as mixed- linked or cereal �-glucans [24].
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Most of the previous studies on immunomodulatory activities of barley dietary fiber have focused on the mixed-linked �-glucans since they are structurally related to fungal and yeast �-glucans that are �-(1�3)-D-glucopyranosyl polymers with �-(1�6) linked side chains. In vitro and in vivo experiments on �-glucan preparations from yeast and fungi have shown immunomodulating properties and a potential to increase host resistance against infections [20]. Mixed-linked �-glucans from barley might have similar effects, although knowledge on immunomodulatory effects of barley polysaccharides is quite limited. Some activities have been reported on commercially available barley �-glucan; Intra peritoneal injections of barley �-glucan into fish enhanced the leukocyte count, phagocytic activity, lysozyme activity, complement activity via the alternative pathway and serum bactericidal activity [25]. Czop and Austen [26] found that turbid preparations of barley �-glucan activate the alternative pathway of the complement system in vitro. In addition, pre-treatment of human monocytes with barley �-glucan inhibited phagocytosis of zymosan particles [26]. �-glucans enhance cytotoxicity of phagocytes or NK cells towards iC3b-opsonized cells by binding to the lectin site on complement receptor 3 (CR3 or CD11b/CD18, Mac-1, �M�2 integrin) and thereby initiate cytotoxic degranulation of NK cells and phagocytosis by other cells [27–29]. Oral administered barley �-glucan increased the efficacy of photodynamic therapy of Lewis lung carcinoma in mice through binding to CR3 [27], but barley �-glucan binds to CR3 with lower affinity than yeast �-glucan [29,30]. Barley �-glucans also enhance the anti-tumor effect of monoclonal antibodies in mice when administered orally [16,31] by being taken up by gastrointestinal macrophages, transported to the spleen, lymph nodes and bone marrow where smaller fragments of glucan are bound to CR3 on granulocytes which in turn kill iC3b-opsonized tumor cells [15]. Barley �-glucan can also bind to Dectin-1[32] and activate NF-�B when Dectin-1, Syk, CARD9 and Bcl10 are co-expressed in the cells [33]. The transcription factor NF-�B plays a critical role in immune, cellular stress and inflammatory responses [34].
Instead of using commercially available dietary fiber from barley in the present study we isolated fiber fraction from barley, both mixed-linked �-glucan and arabinoxylan and tested for immunomodulatory activities related to inflammation. This involved extraction and the use of specific hydrolytic enzymes to isolate pure polysaccharide fractions and determination of biological activities by stimulation of the human colon epithelial cell lines Caco-2 and HT-29 followed by measurement of cell proliferation and cytokine secretion. In addition, we investigated the fiber`s ability to modulate NF-�B activity in monocytes and their influence on the complement system using the complement-fixing test [35], all systems involving factors with relevance to inflammatory processes.
2. Results and Discussion
2.1. Barley Dietary Fiber Fractions
�-glucan and arabinoxylan samples isolated and purified from the common Norwegian barley variety Tyra were the main basis for our investigations. As shown in Table 1, the constituent sugar analysis combined with 1H-NMR [23] (spectra not shown) revealed that WUM-BS contained 70% arabinoxylan and 30% mixed-linked �-glucan. Treatment with lichenase (L) and amyloglucosidase (A) efficiently removed most of the remaining mixed-linked �-glucan from this fraction; the enzyme treated fraction WUM-BS-LA contained 96.4% arabinoxylan. Trace amounts of mannose were
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attributed to the glycoprotein part of the enzyme preparation used. In this fraction, ferulic acid is not present due to alkali treatment during the extraction procedure.
Table 1. Characterization of fiber fractions from Hordeum vulgare var. Tyra: monosaccharide composition (mol %) and estimated molecular weight calculated on the basis of pullulan standards by GPC-SEC and refractive index detection, as weight average (Mw) and number average (Mn) of barley fiber fractions .
Monosaccharide Composition (mol%) Molecular Weight (kDa) Fraction Glc Man Xyl Ara Mw Mn WSM-TP 91.0 0.0 5.8 3.3 1090 599
WSM-TPX 96.5 0.0 1.9 1.6 886 501 WUM-BS 30.0 0.0 52.7 17.3 412 98
WUM-BS-LA 2.0 1.6 66.0 30.4 156 42 WSM-TP was composed of 90% mixed-linked �-glucan in addition to 10% co-extracted
arabinoxylan. Most of the arabinoxylan was removed by enzymatic treatment with xylanase (X). The enzyme treated fraction WSM-TPX was composed of 96.5% glucose and only 3.5% arabinoxylan.
The estimated relative weight average molecular weights (Mw) based on the pullulan series were about 886 and 156 kDa for WSM-TPX and WUM-BS-LA, respectively (Table 1). Molecular weight decreased during the enzymatic treatment of WSM-TP and WUM-BS from about 1090 and 412 kDa, respectively, giving samples with less polydispersities (Mw/Mn).
All previous studies on immunomodulatory activity of mixed-linked �-glucans from barley have been performed on commercially available samples. It should be noticed [20] that choice of isolation method may influence polysaccharide characteristics, such as molecular weight and solubility, and thereby their biological activities. In addition, co-extracted substances or contaminants of an endotoxin nature that may occur during isolation may contribute to significant activities in immunological test systems.
Potential degradation of dietary fiber during food processing has not been taken into account in this study. In addition, the fact that dietary fiber very seldom is eaten alone without subsequent intake of several other food constituents makes the picture quite complex and complicated to explore. In the present study, all fiber fractions had relatively high molecular weight after isolation, and no attempts were made to alter the chain length in either of the samples. This was because we primarily wanted to investigate intact carbohydrate dietary fibers with the presumption that dietary fiber remains undegraded until reaching the microflora in the colon.
2.2. Effect on IL-8 Secretion and Cell Proliferation in Caco-2 and HT-29
To test the inflammatory response of the fiber fractions on gut epithelial cells the modulation of IL-8 (CXCL8) secretion from the human intestinal epithelial cell lines Caco-2 and HT-29 cells was determined. The concentration used of 1 mg/mL is physiologically relevant as a concentration of 1 mg/mL barley fiber in the intestine corresponds to the consumption of approximately 20 g barley, an amount found, for example, in two slices of barley bread (40% barley flour). In addition, the potential toxic effect of the fiber fractions on the Caco-2 and HT-9 cells was determined by measuring the effect of the fiber fractions at different concentrations on cell proliferation using the MTT assay.
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We found that the fiber fractions had no significant effect on the cell proliferation of the human intestinal epithelial cell lines Caco-2 and HT-29 cells (Figure 1A, B). It was observed that the HT-29 cell line in general secretes considerably higher levels of IL-8 than Caco-2, but the barley fiber fractions had no significant effect on this secretion either from Caco-2 (Figure 2A) or HT-29 cells (Figure 2B). Only the positive controls, PMII and zymosan increased secretion of IL-8 from both cell lines significantly (p = 0.001) compared to the respective controls. Zymosan is a crude extract from yeast (Saccharomyces cerevisiae) and contains mainly �-glucan but also some mannan [20], protein, fat and chitin [36]. Immunomodulatory activities of �-glucan from yeast and from other sources have been studied extensively, for review on this topic see [37]. PMII is a pectic polysaccharide fraction isolated from Plantago major L. leaves, a plant used in traditional medicine to aid the healing of wounds. PMII has shown immunomodulatory activities both in vitro and in vivo: Increased resistance against bacterial infection in mice, activation of human monocytes and activation of the complement system [35,38,39]. PMII is therefore considered useful as positive control in immunological test systems.
Even though we did not find any direct effect of the barley fiber fractions on the intestinal epithelial cell model system, barley fiber may affect inflammatory processes and immune response by other mechanisms. As outlined in the introduction, barley fiber may be taken up by intestinal macrophages or M-cells and delivered to underlying immune cells where binding of barley �-glucan to the lectin site of CR3 on effector cells has been shown to enhance cytotoxic activity [15,27].
Figure 1. The effect of fiber fractions extracted from the barley variety Tyra on cell proliferation of (A) Caco-2 cells and (B) HT-29 cells. Cells were incubated with three different concentrations of the respective fiber fraction in cell culture medium for 24 hours before cell proliferation was measured. Each bar represents the mean of at least three experiments performed in triplicate (as % of medium control) ± SD.
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Figure 1. Cont.
Figure 2. IL-8 secretion from Caco-2 (A) and HT-29 (B) cells in response to treatment with fiber fractions extracted from the barley variety Tyra, zymosan and PMII (all 1 mg/mL). Cells were incubated with fiber of the respective fiber fractions in cell culture medium for 24 hours before IL-8 secretion was measured. Each bar represents the average ± SD of one representative experiment from a total of three independent experiments. * p < 0.05.
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Figure 2. Cont.
2.3. The Ability of the Fiber Fractions to Modulate NF-�B Activity in Monocytes
The nuclear transcription factor kappa B (NF-�B) plays a central role in inflammatory response [40]. Thus, to further study the effect of the fiber fractions on the immune response, the ability of the fiber fractions to modulate basal and LPS-induced NF-�B activity was tested using the U937-3�B-LUC monocytic cell line stably transfected with a luciferase reporter containing three NF-�B binding sites. It has been shown that this model system correlates well with in vivo NF-�B activity [41,42] Due to the limitations of the test system, lower concentrations of the samples (0.1, 0.2 and 0.4 mg/mL) were used compared to experiments with the Caco-2 and HT-29 cell lines. However, as the U937-3�B-LUC cell line is quite sensitive, the response is still considered relevant. The activities of the different fiber fractions were compared to the positive control, PMII [39]. Of the different fiber fractions only the highest concentration of WUM-BS had a significant effect on basal NF-�B activity (p = 0.004) (Figure 3A), giving an increase of the activity to 270% compared to control. The apparent dose response from 0.1 mg/mL to 0.4 mg/mL of all fractions of the basal NF-�B activity was statistically not significant compared to the control. However, all concentrations of PMII significantly increased basal NF-�B activity (p < 0.001) in the test system compared to the control. None of the fiber fractions had significant effect on the LPS-induced NF-�B activity, only the highest concentration of PMII increased the LPS-induced NF-�B activity significantly (p = 0.013) (Figure 3B).
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Figure 3. The effect of the fiber fractions extracted from the barley variety Tyra on basal (A) and LPS-induced (B) NF-�B activity. U937-3x�B-LUC cells were incubated with 0.1, 0.2 or 0.4 mg/mL as indicated of the respective fiber fraction in cell culture medium for 6.5 hours before luciferase activity was measured. For LPS-induction, 1 �g/mL LPS was added after 30 min, and the cells incubated further for six hours before the luciferase activity was measured. Each bar represents the mean of at least three experiments performed in triplicate ± SD. * p < 0.05.
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Only high concentrations of a fraction containing 70% arabinoxylan and 30% mixed-linked �-glucan (WUM-BS) increased the activity of the pro-inflammatory transcription factor NF-�B in monocytes. This fraction was obtained after alkaline extraction of a water insoluble residue. Neither pure arabinoxylan nor pure mixed-linked �-glucan was active in this test system. Some biological effects of �-glucans are initiated by binding to Dectin-1 on macrophages and dendritic cells. Barley �-glucan has previously been found to activate NF-�B when Dectin-1, Syk, SARD9 and Bcl10 were co-expressed in the cells [33], and it was concluded that Dectin-1 was involved in these activities. However, binding to Dectin-1 requires �-glucans with a minimum of 10- or 11- mer 1,3-linked glucose oligomers [43] which are structural elements not found in barley. Barley �-glucans only contain single 1,3-linked glucose units separating two or three 1,4-linked glucose oligomers [44]. Transcription factor NF-�B can be activated via many different pathways including proinflammatory cytokines, TLR activation, for example, by LPS and by T-cell activation [40]. As shown in Figure 3B, LPS induced NF-�B activity was not significantly altered by any of the barley fractions indicating that the basal activity observed after stimulation of 0.4 mg/mL WUM-BS may be due to contamination by LPS. In any case, the activity found in Figure 3A cannot be due to either arabinoxylan or mixed-linked �-glucan since other fractions containing higher levels of mixed-linked �-glucan (WSM-TP and WSM-TPX) and arabinoxylan (WUM-BS-LA) were inactive in the test system. The positive control PMII increased LPS induced activity but to a lesser extent than measured with PMII alone (Figure 3A). This shows that PMII is active per se, but confirms presence of LPS. Previously, it has been shown that PMII can activate monocytes and induce secretion of TNF� [39]. One might speculate that secreted TNF� in turn activate NF-�B [40], alternatively PMII may bind to NF-�B activating receptors directly.
2.4. Complement Fixing Test
Purified �-glucan (WSM-TPX) and arabinoxylan (WUM-BS-LA) from Tyra were tested for activity in the complement-fixing test. Both showed lower activities than the positive control, PMII. At 1 mg/mL WSM-TPX was significantly more active than WUM-BS-LA (p = 0.009) (Figure 4).
�-glucans and purified arabinoxylans from other barley varieties [45] were also subjected to this test. As shown in Figure 4, all arabinoxylan fractions (WUM-BS-LA) had relatively low activity compared to the positive control. Arabinoxylan isolated from Tyra, NK96300 and SB94897 had very similar activities; the one from CDC Gainer was almost inactive.
Starch-free mixed-linked �-glucans that had not been subjected to a xylanase treatment (WSM-TP) containing additional small amounts of arabinoxylan, had the highest activity in this test system. Such WSM-TP samples originating from CDC Gainer and NK96300 showed activity at the same level as the positive control, while similar fractions from Tyra and SB94897 were less active (p < 0.035). Figure 4 furthermore shows that a xylanase purification step of WSM-TP into purified mixed-linked �-glucan (WSM-TPX) did not alter the complement-fixing activity significantly.
In general, all mixed-linked �-glucan rich fractions had a significantly higher complement-fixing activity than the arabinoxylan-rich fractions (p < 0.027).
According to Figure 4 the fractions can be listed as follows with regard to decreasing activity in the complement fixing test: PMII = WSM-TP CDC Gainer = WSM-TP NK96300 > WSM-TP Tyra =
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WSM TPX Tyra = WSM TP SB94897 > WUM BS-LA Tyra = WUM-BS-LA NK96300 = WUM-BS-LA SB94897 > WUM-BS-LA CDC Gainer.
Figure 4. Complement fixing test of fiber fractions isolated from barley varieties. Each bar represents % activity (mean values of triplicates ± SD) of the positive control PMII measured at 1 mg/mL. The fractions are arabinoxylan (WUM-BS-LA) and mixed-linked �-glucan rich fractions (WSM-TP and WSM-TPX) from the barley varieties Tyra, NK96300, SB94897 and CDC Gainer. Activity bars denoted with the same letter (a, b or c) are not significantly different, p < 0.05.
Mixed-linked �-glucans from barley have previously shown to activate the complement system via the alternative pathway [26], the present study demonstrates an effect also on the classical pathway. The complement system provides a first line of protection against potential harmful invaders and is part of the innate immune system. It consists of a group of serum proteins that are activated in a cascade mechanism. Many of these proteins are pro-enzymes that are activated by proteolytical cleavage which in turn activate the next step in the cascade. Activation can be initiated by three pathways; the classical pathway, the alternative pathway or the lectin pathway, and is important for initiating inflammation, activation of leucocytes, lysis of target cells and opsonisation [46,47]. The test system employed has some limitations since it does not distinguish between activation and inhibition of the complement cascade, only a “consumption” of complement activity is registered. From previous studies however, it is established that PMII, the positive control, is an activator of the complement system [35], and it has also shown to protect against bacterial infection in vivo [38].
The mixed-linked �-glucan fractions tested were more active than the arabinoxylan fractions. The reason for the differences in activity of the different �-glucans might be due to differences in their primary structure. The ratio of (1�4)/(1�3) linkages present varies between the different barley varieties tested. NK96300 has the highest ratio (2.76) followed by CDC Gainer (2.59), Tyra (2.48) and SB94897 (2.30) [23]. The varieties with the highest (1�4)/(1�3) ratio have the highest activity in the complement fixing test, but statistical analysis shows no significant correlation between linkage ratio and activity or between molecular weight of the WSM-TP fractions and activity. Mw of WSM-TP
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fractions from NK96300, CDC Gainer and SB94897 were estimated to 1040, 1130 and 1040 kDa, respectively. The estimated molecular weights of the WSM-TP fractions were significantly higher than the corresponding arabinoxylan (WUM-BS-LA) fractions (p < 0.001). Mw of the WUM-BS-LA fractions from NK96300, CDC Gainer and SB94897 were estimated to 214, 203 and 190 kDa, respectively. The WUM-BS-LA fractions from different barley varieties tested had in general low activities in the complement fixing test. However, when both these arabinoxylan fractions and the more active �-glucan rich fractions were included in the statistical test a significant positive correlation was found between molecular weight and activity (p = 0.002). On the other hand, when these two classes of dietary fiber are evaluated separately there is no correlation between activity and their estimated molecular weights. Contamination of LPS does not affect this test system [48], so activity is not attributed to presence of endotoxin.
The arabinoxylans from different barley varieties tested had in general low activities in the complement fixing test leaving this hemicellulose type of dietary fiber non-responsive in all the test systems in the present study. To our knowledge, high molecular weight arabinoxylans have not been ascribed immunomodulatory activities. On the other hand arabinoxylan oligosaccharides have been studied for prebiotic properties [49] and have been shown to reduce preneoplastic lesions in the colon of rats treated with a carcinogen [50].
3. Experimental Section
3.1. Isolation of Fiber Fractions
Fiber fractions were extracted from four barley varieties; NK96300, Tyra, CDC Gainer and SB94897 basically as previously described [51]. Briefly; milled (0.5 mm) barley samples (48 g) were extracted and washed with boiling ethanol. This removed low molecular weight constituents and is promoting the denaturation of endogenous hydrolytic enzymes such as �-glucanase. Following defatting with hexane, extraction with boiling water gave a water soluble material (WSM) and a residue of water insoluble material (WUM). WSM was furthermore treated with 7 mL amylase (Termamyl 120 L, Type L, Novozymes ) and 75 mg protease (Porcine Pancreatine, SIGMA) filtered and recovered with alcohol precipitation resulting in the starch free fraction designated WSM-TP.
In an attempt to remove small amounts of co-extracted arabinoxylan, WSM-TP was treated with a xylanase. WSM-TP Tyra (1 g) was dissolved in sodium acetate buffer pH 4.5, and 10 μL (21 U) endo �-xylanase (�-xylanase M6, Megazyme) was added at 40 °C and left for 3 h with gentle stirring. Polysaccharide material was precipitated with isopropanol and centrifugated at 1000 × g for 10 min. The pellet was redissolved in water, dialyzed against distilled water using a dialyzing tube with cut off 12,000–14,000 (Medicell Int. Ltd); freeze dried and designated WSM-TPX.
Base soluble material (WUM-BS) was then extracted from the previous water insoluble residue (WUM) with 1 M NaOH added 1% NaBH4. Co-extracted mixed-linked �-glucans and starch were removed by adding 50 U lichenase (Lichenase EC 3.2.1.73, Megazyme) and 400 μL amyloglucosidase (Amyloglucosidase for Total Dietary Fiber Assay EC 3.2.1.3, SIGMA) as described elsewhere [51] giving fractions designated WUM-BS-LA.
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3.2. Monosaccharide Composition
Methanolysis combined with TMS-derivatisation and GC were performed according to the method of Chambers & Clamp [52] with modifications as previously described [53] using 4 M HCl in anhydrous methanol for 24 h at 80 °C.
3.3. 1H-NMR
1H-NMR spectra of selected samples were obtained on a Varian Mercury 300 system. Approximately 4 mg of freeze dried material was solubilized in 0.7 mL D2O, transferred to NMR glass tubes and acquired at 80 °C with typically 64 scans. Further details of the method are described in Knutsen & Holtekjolen [23] .
3.4. HPLC
GPC-SEC was performed using a DIONEX P680 pump with a Spectraphysics AS3500 auto injector and a Shimadzu RID6A refractive index detector controlled with Chromeleon 6.80 software. Serially connected Shodex OHPack SB-806-HQ and SB 804-HQ columns were connected to a Shodex OHPack SB-LG precolumn and eluted at 40 °C with 50 mM Na2SO4 (0.5 mL/min), and samples (1 mg/mL) were injected using a 100 μL loop. Relative molecular weight averages (Mw and Mn) were estimated offline by the software WINGPC �6.2 using pullulan molecular weight standards ranging from Mp 342 to 1,520,000 Da for calibration. Software and standards were obtained from PSS (Polymer Standards Service GmbH, Mainz, Germany).
3.5. Cell Cultures
The Caco-2 cell line (obtained from the American Type Culture Collection (ATCC), and a generous gift from Professor Kirsten Sandvig, Norwegian Radium Hospital) and HT-29 cell line (obtained from ATCC, and a generous gift from Professor Tor Lea, Norwegian University of Life Sciences) were grown in DMEM medium containing 10% fetal calf serum, 1% non-essential amino acids, 100 U/mL penicillin, and 100 mg/mL streptomycin. The U937-3xkB-LUC cell line (a generous gift from Professor Rune Blomhoff, University of Oslo) was grown in RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 50 U/mL penicillin, 50 mg/mL streptomycin and 75 μg/mL hygromycin (Sigma-Aldrich, St. Louis, MO). The cells were maintained at 37 °C and 5% CO2 in a humidified incubator. If not otherwise stated, all solutions were obtained from Invitrogen (Carlstad, CA).
3.6. Measurement of Cell Proliferation and IL-8 Secretion
For IL-8 secretion, cells were plated in 12-well plates. For cell proliferation, cells were plated in 96-well plates. Cells were plated at a concentration of 1.0 × 105 cells/mL (Caco-2) and 1.5 × 105 cells/mL (HT-29) and incubated until they reached 80 % confluency (48 h). The fiber fractions were solubilized in water by boiling for 20 min, aliquoted, freeze dried and re-solubilized in growth medium to treatment concentrations of 0.5–3 mg/mL. A yeast derived beta-glucan (Zymosan A
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Z4250 Sigma) and PMII, a plant polysaccharide fraction from Plantago major L with known immune stimulating activity [35,38,39] were used as positive controls for IL-8 secretion. Cells were incubated 24 h with 1.5 mL (12-well plates) or 100 μL (96-well plates) growth medium or solubilized fiber fractions in duplicate (12-well plates) or triplicate (96-well plates). At the end of the incubation, the plates were processed for measurement of either cell proliferation or IL-8 secretion.
Cell proliferation was determined using the colorimetric MTT assay (Roche Diagnostics GmbH, Mannheim, Germany) that measures the ability of metabolic active cells to cleave tetrazolium sodium salt to purple formazan crystals [54]. The resulting purple precipitate in each cell was dissolved in 100 �L isopropanol containing 0.04 M HCl, and the absorbance measured at 562 nm using Titertek Multiscan plus MK II plate reader (Labsystems, Finland). IL-8 concentrations in the cell culture supernatants were determined using an enzyme linked immunosorbent assay (ELISA). Monoclonal mouse anti-human IL-8 antibody (BD Bioscience Pharmingen, San Diego, CA) suspended in coating buffer (0.1 M Carbonate/Bicarbonate buffer pH 9.6) was added to MaxiSorpTM ELISA plates (Nunc, Roskilde, Denmark) and incubated over night at 4 °C. Plates were washed three times with PBS containing 0.01% Tween-20 and unspecific binding-sites were blocked by incubating with 5% BSA in PBS for 1 h at room temperature. After washing five times with PBS-Tween, samples and human recombinant IL-8 standards (BD Bioscience Pharmingen,) diluted in working strength high performance ELISA (HPE) buffer from Sanquin (Amsterdam, Netherlands) were added to the plates, which were then incubated for 1.5 h at room temperature followed by washing five times with PBS-Tween. Plates were then incubated for 1 h with biotinylated mouse anti-human IL-8 monoclonal antibody (BD Bioscience Pharmingen) in HPE buffer. After another washing step streptavidin-horseradishperoxidase conjugate (BD Bioscience Pharmingen) in HPE buffer was added and incubated at room temperature for 30 min. Plates were then washed five times with 30 sec between each wash. Color developed after addition of 3,3’,5,5’-tetramethylbenzidine (Sigma-Aldrich) in 0.05 M Phosphate-Citrate-Buffer containing H2O2. After 10 min the reaction was stopped by addition of 1 M H2SO4, and absorbance was measured at 450 nm using the Titertek Multiscan plus MK II plate reader (Labsystems, Finland). The detection limit of the IL-8 ELISA was 2 pg/mL.
3.7. NF-kB Activity Assay
In order to measure NF-�B activity the U937-3x�B-LUC cell line were transferred to RPMI medium with 2 % fetal bovine serum and seeded out in 96 well plates. The fiber fractions extracted from the barley variety Tyra were mixed with highly purified water (Milli-Q, 18.2 M� to a final concentration of 1 mg/mL in Precellys CK14 homogenization tubes (Bertin Technologies, Montigny le Bretonneux, France), solubilized using the Precellys 24 homogenizer (Bertin Technologies) followed by boiling the samples for 5 min, and then freeze dried. The resulting freeze dried fiber fractions were dissolved in medium with 2% serum to a concentration of 4 mg/mL. PMII from Plantago major L. leaves was used as positive control [35,38,39]. This final solution of the fiber fractions was diluted directly in the wells giving the final concentrations of 0.1, 0.2 and 0.4 mg/mL. To measure basal NF-�B activity, cells were incubated with fiber fractions or vehicle control for 6.5 hours. To measure lipopolysaccharide (LPS)-induced NF-�B activity, cells were pre-incubated with fiber fractions or vehicle control for 30 min, then 1 μg/mL lipopolysaccharide isolated from E. coli 0111:B4
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(Sigma-Aldrich, St. Louis, MO, U.S.) was added to the cells and the incubation continued for 6 hours. Cell viability for these cells was determined with the use of the CellTiter-Glo Luminiscent Cell Viability Assay (Promega, Madison, WI, U.S.) with cut-off value of 10% non-viable cells. The NF-�B activity was determined by measuring the luciferase activity after addition of Bright-GloTM Reagent (Pomega, Madison, WI, U.S.) in accordance to the manufacturer’s instructions. Luminescence was detected for 1 sec using the Glomax96 Microplate Luminometer (Promega, Madison, WI, U.S.).
3.8. Complement Fixing Test
Human complement proteins were incubated with fiber fractions that might either activate or inhibit activation of the complement proteins. In both situations complement activity is depleted with a negative influence on a balanced hemolysis system involving antibody-sensitized sheep red blood cells and a human serum diluted to give 50% hemolysis. The degree of hemolysis was measured as absorbency at 405 nm. Fiber fractions were tested in triplicates using PMII, a polysaccharide fraction from Plantago major L. as positive control [35,39].
3.9. Statistics
Analysis of significant differences was tested by one-way analysis of variance (ANOVA) with Dunnett’s comparisons with a control and Pearson correlation analysis using Minitab Version 16. Differences were considered significant when p < 0.05.
4. Conclusions
From the experiments presented, it is concluded that purified high molecular weight mixed-linked �-glucans from barley have quite low immunological responses and do not affect proliferation and secretion of IL-8 of the colon epithelial cell lines Caco-2 and HT-29, or NF-kappaB activity in the monocytic cell line U937-3�B-LUC but are active in the complement-fixing test. High molecular weight barley arabinoxylans have neglectible activities in all test systems mentioned. Taken together the results do not support that barley dietary fiber protect against the development of CRC through the immune responses or inflammatory responses tested. Still, one cannot overrule that such effects may occur through other mechanisms that may be shown in other test systems.
Acknowledgements
The project was financed by funding from Infigut (NFR 185125). Novozymes Nordic is thanked for the supply of Termamyl. Ann-Katrin Holtekjølen is thanked for providing the fiber fractions from different barley varieties. The authors acknowledge Merete Rusås Jensen, Nofima Mat, Norwegian Institute of Food Fisheries and Aquaculture Research, for technical assistance.
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