University of Groningen Innate and Adaptive Immune Effects of Chicory

University of Groningen

Innate and adaptive immune effects of chicory root dietary fibers Vogt, Leonie

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Innate and adaptive immune effects of chicory root dietary fibers

L.M. Vogt

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Publication of this thesis was financially supported by: GUIDE, University Medical Center Groningen and University of Groningen.

Innate and adaptive immune effects of chicory root dietary fibers

Proefschrift

ter verkrijging van de graad van doctor aan de Rijksuniversiteit Groningen op gezag van de rector magnificus prof. dr. E. Sterken en volgens besluit van het College voor Promoties.

De openbare verdediging zal plaatsvinden op

maandag 12 oktober 2015 om 14.30 uur

door

Leonie Marloes Vogt

geboren op 18 augustus 1983 te Delfzijl

Promotor Prof. dr. P. de Vos

Copromotor Dr. M.M. Faas

Beoordelingscommissie Prof. dr. H.A. Schols Prof. dr. L. Dijkhuizen Prof. dr. ir. W. van den Ende

Paranimfen Marlies Elderman Neha Sahasrabudhe

Voor pap en mam

Table of contents

Chapter 1 1 General introduction

Chapter 2 53 Immune modulation by different types of β2→1-fructans is Toll-like receptor dependent Chapter 3 77 Toll-like receptor 2 activation by β2→1 fructans protects barrier function of T84 human intestinal epithelial cells in chain length-dependent manner Chapter 4 97 Cellulose alters the expression of NF-κB-related genes and TLR-related genes in human peripheral blood mononuclear cells Chapter 5 123 The impact of lemon pectin characteristics on TLR activation and T84 intestinal epithelial barrier function Chapter 6 147 Long chain inulin-type fructans but not short chain inulin-type fructans enhance hepatitis B vaccination response in young adults Chapter 7 175 General discussion Chapter 8 191 General summary Nederlandse samenvatting 199 Dankwoord 207 APPENDICES 215

CHAPTER 1

General Introduction and scope of the thesis

Immunological properties of inulin-type fructans

L.M. Vogt1, D. Meyer2, G. Pullens3, M.M. Faas1,4, M.J. Smelt1, K. Venema5, U. Ramasamy6, H.A. Schols6, P. de Vos1

1 Department of Pathology and Medical Biology, Division Medical Biology, Groningen University, University Medical Center Groningen 2 Sensus B.V., Borchwerf 3, 4704 RG Roosendaal, The Netherlands 3 Cosun Food Technology Centre, Oostelijke Havendijk 15, 4704 RA, Roosendaal, NL 4 Department of Obstetrics and Gynaecology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, the Netherlands 5TNO Quality of Life, Department of Biosciences, PO Box 360, 3700 AJ Zeist, The Netherlands 6Laboratory of Food Chemistry, Wageningen University, PO Box 8129, 6700 EV Wageningen, The Netherlands

Published in modified form in Crit Rev Food Sci Nutr., vol. 55, p. 414-436, Febr. 2015.

CHAPTER 1. IMMUNOLOGICAL PROPERTIES OF INULIN-TYPE FRUCTANS.

Abstract

Beneficial effects of inulin-type fructans are discussed in view of studies that applied the oligosaccharides in colon cancer, chronic inflammatory diseases, vaccination efficacy, and prevention of infection and allergy. In this chapter, we discuss their immunomodulating effects. It is suggested that immunomodulation is elicited through indirect and direct mechanisms. Indirect mechanisms encompass stimulation of growth and activity of lactic acid bacteria, but can also be caused by fermentation products of these bacteria, i.e., short chain fatty acids. Evidence for direct effects on the immune system generally remains to be confirmed. It is suggested that inulin-type fructans can be detected by gut dendritic cells (DCs), through receptor ligation of pathogen recognition receptors (PRRs) such as Toll-like receptors, nucleotide oligomerization domain containing proteins (NODs), C-type lectin receptors, and galectins, eventually inducing pro- and anti-inflammatory cytokines. DCs may also exert antigen presenting capacity toward effector cells, such as B cells, T cells, and natural killer cells locally, or in the spleen. Inulin-type fructans may also ligate PRRs expressed on gut epithelium, which could influence its barrier function. Inulin-type fructans are potent immunomodulating food components that hold many promises for prevention of disease. From this literature review, we conclude that studies into the mechanisms, dose- effect relations, and structure-function studies are warranted. Inulin-type fructans from chicory can then be applied as model fibers to establish a technology platform for testing dietary fibers in primary cells and cell line models, and finally to test the application in human studies.

CHAPTER 1. IMMUNOLOGICAL PROPERTIES OF INULIN-TYPE FRUCTANS.

Introduction

1.1 Functional food fibers in the context of society

The scientific and industrial functional food worlds are meeting some new challenges. The consumer awareness that food is not only required to supply energy and nutrition, but also that healthy food is essential for prevention of disease and for both physical and mental well-being is growing [1-3]. This causes an increased demand for functional foods. A major category of functional foods is that of dietary fibers and prebiotic fibers. One of the root vegetables which is specifically cultured for its high content of prebiotic inulin-type fructan fibers is Cichorium intybus, or common chicory [4]. Inulin-type fructans are naturally occurring linear plant oligo- and polysaccharides which consist of minimally two fructose-units, and at least one β(2-1) fructosyl-fructose glycosydic bond [4]. It is a family of molecules which meet the three classification criteria for being considered a prebiotic, as defined by Gibson and Roberfroid [5]; i.e. resistance to hydrolysis or absorption in the upper gastrointestinal (GI) tract, fermentation by the intestinal microbiota, and selective stimulation of the growth and/or activity of beneficial intestinal bacteria, such as Lactobacillus species and Bifidobacterium species. Well-known effects of inulin-type fructans on the gut microbiota are the increase in numbers of these types of bacteria in the intestinal tract, and the selective fermentation of inulin-type fructans by most Bifidobacterium species [6], and by some Lactobacillus species [7]. For a considerable period of time, research has mainly been focused on the prebiotic, i.e. indirect effects of inulin-type fructans [8-12]. Somewhat more recent is the notion that prebiotic carbohydrates such as inulin-type fructans may elicit additional, direct effects such as immunomodulation along the GI tract [5,8,11,13,14]. This may occur via direct contact with gut dendritic cells (DCs) which sample immune active components from the gut lumen, and with intraepithelial lymphocytes (IELs) which can respond immediately upon contact with immune active food components [14-16]. It is also conceivable that contact of inulin-type fructans with the gut epithelial cells modulates the innate immune barrier by modifying epithelial tight junction integrity, or alters the signals from epithelial cells to the underlying immune cells [17]. In addition, the glycosidic and non-glycosidic fermentation products produced by gut

CHAPTER 1. IMMUNOLOGICAL PROPERTIES OF INULIN-TYPE FRUCTANS. microbiota upon fiber supplementation are under investigation for their beneficial health effects as reviewed by Meijer et al. [18] and Macfarlane and Macfarlane [19]. The glycosidic fermentation products can be small sized oligosaccharides and the non-glycosidic fermentation products include SCFAs such as acetate, propionate, and butyrate [20,21]. Although there are some recent reviews on the immunomodulatory properties of inulin-type fructans [22-24], in the majority of these reviews, immunomodulation was mainly discussed as an integral part of the health benefits of prebiotic fibers [11,25-28]. Many studies demonstrate that inulin-type fructans have unique ways for immunomodulation, but the underlying mechanisms are still incompletely understood. The following section provides an overview of the current knowledge of the direct and indirect mechanisms of immune modulation by inulin-type fructans and the possible signaling pathways. This overview is focused on the beneficial effects of inulin-type fructan supplementation in several diseases including colon cancer, [12,29,30,30,31], chronic inflammatory diseases [32-36], vaccination efficacy [37,38,38-43], and prevention of infection and allergy [8,39,44-50].

1.2 Structure and terminology of inulin-type fructans Before discussing the immunomodulating properties of inulin-type fructans it is mandatory to discuss the structure and terminology of these molecules since their structure or more specifically their chain length probably determines their function in the host. Several studies have indicated that polymer chain length or degree of polymerization (DP), is an important feature to consider, as it determines where along the GI tract fermentation occurs [9,51,52]. It appears that short chain fructans are generally fermented relatively fast in the proximal colon, whereas fructans with a relatively long chain resist fermentation until they reach the distal colon where they are metabolized [10,17]. In addition, Bifidobacterium species differ along the gastrointestinal tract, so the different DP can determine the types of bacteria that become enriched. This could render different outcomes in health related parameters [9]. Fructans are denoted as Fn, with F for fructose and n representing the number of fructose subunits in the polymer. Most inulin-type fructans in nature contain a terminal glucose residue (denoted as a GFn) as biosynthesis starts with sucrose to which fructose residues are added [4]. Figure 1 depicts Haworth projections of these two types of fructans. When

CHAPTER 1. IMMUNOLOGICAL PROPERTIES OF INULIN-TYPE FRUCTANS.

Figure 1. Haworth projections of fructan molecules. Left projection depicts an inulin-type fructan of the GFn type, right projection depicts an inulin-type fructan of the Fn type. the fructan chain starts with a glucose molecule, this glucose can be removed from the chain by hydrolyzing sucrase enzymes, which are produced at the tips of the small intestinal epithelial villi [53]. Based on chain length, inulin-type fructans are usually rather arbitrarily divided in subcategories with a relatively small (2 to 4), medium (5 to 10) and relatively large chain length (11 to 60 fructose units). Over the course of time the nomenclature to describe inulin-type prebiotics has been inconsistent. Historically, the term fructooligosaccharides or FOS was used for DP 3-5 material derived from sucrose which is thereby only of the GFn type [54]. The term oligofructose or OF was used for DP3-10 material derived from native inulin which can be of both the GFn and the Fn type [55]. Later, FOS and OF were and are more and more used as synonyms to describe fructans with a chain length ranging between 2 and 10 subunits [56]. To discriminate, the term short chain FOS (sc-FOS) was used by the company producing this ingredient (Actilight®, Eridania-Beghin Say, Belgium), [57]. Some companies use the term long chain FOS (lc-FOS) or OF (lc-OF) for the long chain inulin that is part of a specific galactooligosaccharide (GOS)/inulin mixture. The term inulin is often applied to inulin-type fructans with chain lengths above 10 subunits,

CHAPTER 1. IMMUNOLOGICAL PROPERTIES OF INULIN-TYPE FRUCTANS.

Figure 2. Chain length distribution examples of inulin-type fructans for FOS, FOS-enriched inulin, and high average molecular weight inulin. however, inulin is the generic term describing all β(2,1)-fructans without specification of chain length [4,11]. Here we will apply the term FOS for inulin-type fructans of 2 to 10 subunits and we will use the term inulin for inulin-type fructans with chain lengths above 10 subunits. Chain length is specified where possible to render an overview of the properties of these specific compounds. Figure 2 depicts chain length profiles as an example for a FOS, a FOS-enriched inulin and a high average molecular weight inulin.

1.3 The gastrointestinal immune barrier and inulin-type fructans Many of the studies addressing immunomodulating effects of inulin-type fructans have focused on the Gut Associated Lymphoid Tissue (GALT, Figure 3). Constituents of this tissue are the lamina propria, Peyer’s patches with follicles containing B and T lymphocytes, isolated lymph nodes, mesenteric lymph nodes, and the appendix [58]. Important players in this system are follicle associated Microfold cells (M cells), which are part of the epithelial layer covering the Peyer’s patches, and are specialized in transporting antigens from the lumen to the GALT [59]. Dendritic cells (DCs) and intraepithelial lymphocytes (IELs) lie in between and just below the epithelial surface. The DCs are capable of sampling and sensing the events in the gut lumen and are strong antigen presenting cells (APCs) [60]. Lamina propria DCs can respond to antigens which have

CHAPTER 1. IMMUNOLOGICAL PROPERTIES OF INULIN-TYPE FRUCTANS.

Figure 3. Gut-associated lymphoid tissue (GALT). Schematic representation of the GALT structure; (a) Gut lumen. (b) Lamina propria. (c) Enterocyte lining. (d) Peyer’s patch. (e) Microfold cell. (f) Follicle with B and T lymphocytes. (g) Mesenteric lymph node. (h) Lamina propria mast cell. (i) Lamina propria lymphocyte. (j) Dendritic cell penetrating enterocyte monolayer and sampling gut lumen. (k) Intraepithelial lymphocyte. (l) Lymphoid aggregates. (m) High endothelial vessel. N.B. Structural proportions were altered for illustrative purposes. penetrated gut tissue beyond the epithelial barrier and are also strong APCs [60]. Depending on the cytokine environment, APCs can determine whether the T cells they encounter and present their antigen to, differentiate into regulatory T lymphocytes (Tr1 or Th3) or into effector (helper) T lymphocytes (Th1, Th2, or Th17) [59]. Antigen presentation can occur in the lamina propria or specifically in Peyer’s patches or mesenteric lymph nodes [59]. The immunoglobulin (Ig) M+ B lymphocytes in Peyer’s patch follicles are plasma-cell precursors that produce IgA. Memory IgA+ B lymphocytes are generated in the germinal centers of these follicles [59,61,62]. IgA is mainly synthesized in response to T-lymphocyte activation and the production is again regulated by the cytokine environment. Interleukin (IL)-5, IL-6, and IL-10 stimulate final differentiation of B lymphocytes into IgA-secreting plasma cells [63]. IgA is the most abundant immunoglobulin in the intestinal mucosa (80-90%) and forms the first line of defence against colonization and invasion by pathogens, and against damaging toxins [64]. T lymphocyte subtypes can

CHAPTER 1. IMMUNOLOGICAL PROPERTIES OF INULIN-TYPE FRUCTANS. be characterized by the cytokines they produce. Th1 lymphocytes typically secrete Interferon (IFN)-γ, IL-2, and Tumor Necrosis Factor (TNF)-β, and their main function is phagocyte-mediated defense against viral, bacterial, and protozoic infections. Th2 lymphocytes typically secrete IL-4, IL-5, and IL-13, and act as allergic response mediators and defenders against infections produced by helminths and arthropods [63,65]. Although it is becoming clear that the Th1/Th2 model is too simplistic, the Th model has still played an important part in developing our understanding of the roles and behavior of Th cells and the cytokines they produce during an immune response. Therefore, other subtypes to discuss are Th3 cells, Th5 cells, Th9 cells, Th17 cells, and Th22 cells. Th3 cells produce the cytokine Transforming Growth Factor-beta (TGF-β) and IL-10. Both cytokines are inhibitory to Th cells; TGF-β suppresses the activity of most of the immune system. Th5 cells constitute a subpopulation of Th cells described by Kurowska-Stolarska et al. [66]. These cells produce mainly IL-5, but not IL- 4, both of which are characteristic type 2 cytokines produced by Th2 cells. Studies by Veldhoen et al. [67] revealed that another Th subset may exist. Th9 cells are claimed to be an IL-9 producing T cell subset focused on defending helminth infections. These cells have been identified as a unique subset of Th cells and constitute a subset of cells known as neutrophil-regulatory T-cells. They are CD4(+) T-cells that are defined by the production of IL-17. Th17 cells develop from naive T-cells along a pathway that is distinct from the differentiation pathways that give rise to the Th cell populations known as Th1 cells and Th2 cells [68,69]. Th22 cells are IL-22-producing cells which coexpress the chemokine receptor CCR6 and the skin-homing receptors CCR4 and CCR10. This subset of IL-22- producing cells is suggested to be important in skin homeostasis and pathology [70]. All these different arms of the gastrointestinal immune barrier can be modulated either indirectly, i.e. via microbiota or directly upon consumption of inulin-type fructans.

1.4 Indirect mechanism of immunomodulation: Bifidobacteria and SCFAs The prebiotic effects of inulin-type fructans are often referred to as bifidogenic effects. These bifidogenic effects were shown in infants [50,71- 74], adults [75-79], and elderly [57,80,81]. Classically the beneficial effect of inulin-type fructans was assumed to be determined by the effects on the commensal microbiota that formed a barrier for pathogens to enter the host. However, its beneficial effect on commensals probably also has an effect on prevention of inflammation in the systemic circulation. The

CHAPTER 1. IMMUNOLOGICAL PROPERTIES OF INULIN-TYPE FRUCTANS.

fermentation products of inulin-type fructans are carbondioxide, hydrogen, lactate, and SCFAs, including acetate, propionate, and butyrate [4]. These products have been studied in relation to beneficial effects and have been reported to protect from colonization by pathogens or non- commensals by acidification of the colonic content [9,51,82]. In addition, they are rapidly adsorbed by the human body [83] and exert their effect on immune cells by binding to activating G protein-coupled receptors (GPR) [84] GPR41 and GPR43 [84-86]. GPR43 is highly expressed in polymorphonuclear cells (PMNs, e.g. neutrophils) and at lower levels in Peripheral Blood Mononuclear Cells (PBMCs) and purified monocytes. GPR41 is similarly expressed in PBMCs but not in PMNs, monocytes and DCs [86]. Both receptors are equally expressed in bone marrow and spleen. The possible immunomodulatory functions of SCFAs are highlighted by a study in GPR43-/- mice [87]. These mice suffer more from inflammation due to lack of GRP43 binding by SCFA, which normally results in anti-inflammatory effects [18]. In these mice, production of inflammatory mediators and immune cell recruitment are increased. These results suggest an immunoregulatory effect for SCFA-mediated GPR43 signaling. More studies are required to confirm whether inulin-type fructan supplementation and subsequent SCFA production actually affects these receptors, but as it has a strong effect on SCFA producing bacteria it is very likely a mechanism by which inulin-type fructans exert their immunomodulatory effect [88]. The intraindividual bifidogenic effect can differ in outcome depending on the initial level of bifidobacteria, and may also differ between individual Bifidobacterium species [9]. Although in general this prebiotic effect is present [73,75,76,79,89] there are inconsistencies in prebiotic properties of inulin-type fructans throughout literature [4]. These should probably be explained by differences in the applied type i.e. chain length of fructan, the dose, the study population, the duration of supplementation, and the time intervals for microbiological analysis [4,71,73,76,79]. The digestible mono-, and dimers of fructose or glucose which are present in most prebiotics may also influence the bacterial composition upon supplementation. Minor data are available on possible differences in effects of chain length of inulin-type fructans on prebiotic effects, besides the fact that short chain fructans are fermented by more Bifidobacterium species compared to long chain fructans [90].

CHAPTER 1. IMMUNOLOGICAL PROPERTIES OF INULIN-TYPE FRUCTANS.

1.5 Direct mechanisms of immunomodulation: ligation of Pattern Recognition Receptors To be able to distinguish the good from the bad, the gut immune system is equipped with pattern recognition receptors (PRRs). These PRRs recognize molecular structures that are highly conserved and broadly shared by pathogens, known as pathogen-associated molecular patterns (PAMPs) [91]. PRRs include the well-known Toll-like receptors (TLRs), membrane- bound C-type lectin receptors (CLRs), cytosolic proteins such as NOD-like receptors (NLRs) and RIG-I-like receptors (RLRs), and still to be discovered PRRs that mediate sensing of cytosolic DNA or retrovirus infection [92-94]. Upon PAMP recognition, PRRs initiate signaling processes that may lead to cytokine release, inflammation, and clearance of the pathogen as a final goal. Possible direct effects of inulin-type fructans are thought to entail ligation of PRRs on the surface of gut DCs which continuously sample the gut content and are strong APCs [60]. Potential receptors involved are TLRs [95]. TLRs are involved in epithelial cell proliferation, secretion of IgA into the gut lumen and expression of antimicrobial peptides, which are crucial factors for maintaining a healthy epithelial barrier [96,97]. They are typically known to possess carbohydrate binding properties and upon ligation will instigate several immune responses. For the same reasons, CLRs, NLRs, and galectins are also potentially involved in inulin-type fructan signaling [95]. Besides DCs, many cell types express TLRs, including epithelial cells [96]. It is conceivable that as polysaccharides, inulin-type fructans could ligate TLRs on the gut epithelial cells and thereby modulate barrier function by promoting tight junction stability, similar to the mechanism reported by Karczewski et al. [98]. In addition, the activation of epithelial TLRs could alter their interactions with or signals towards surrounding immune cells such as DCs or IELs [16]. Finally, inulin-type fructans may possess the capacity of interacting with cell membrane lipids or even inserting in the membrane [99]. Vereyken et al. [100-102] found that there was a chain length-dependent interaction of inulin with lipids, and that inulin-type fructans can actually interact with or even insert into membrane lipid bilayers [100-102]. This could have a consequence for stimulating events; if insertion renders the membrane more fluid and more dynamic this may facilitate or enhance receptor clustering and subsequent signal transduction. Notably, this may be more or only relevant for sites where the mucus layer is relatively thin i.e. the small bowel [103], so fructans can

CHAPTER 1. IMMUNOLOGICAL PROPERTIES OF INULIN-TYPE FRUCTANS.

reach the cells relatively easily as compared to the large bowel, where the mucus layer is considerably thicker. It should be noted that most hypotheses on direct ligation of inulin-type fructans or their direct contact with epithelial structures remain to be investigated and are at this point still only speculative.

1.6 Experimental evidence for immunomodulation Many supplementation studies with inulin-type fructans were performed in healthy experimental animal models. Some studies apply inulin-type fructans only in a synbiotic treatment, i.e. in combination with a probiotic, limiting the possibility to evaluate the actual fructan effects. The features reported most frequently in healthy experimental animals are increased IgA secretion in serum and fecal samples, and increased IL-10 and IFN-γ production in two specific structures of the GALT; the mesenteric lymph nodes and the Peyer’s patches [104-107] (Table 1). Inconsistency in results are present for IgA in ileum, serum, and feces [104-106,108,109]. The IgA production was increased or not affected [104-106,108,109], the number of lymphocytes in the blood was increased or unaltered [64,106,107,110,111], and the number of lymphocytes or subsets in the spleen and thymus were increased or unaltered [51,64,72,112]. In a single study in sea bream, inulin-type fructan supplementation significantly inhibited phagocytosis and respiratory burst in lymphocytes [113]. However, in a study in salmon, supplementation with 7,5% inulin did not protect against soybean meal-induced colitis [114]. Evidence for immunomodulation on a genetic level was reported by Yasuda et al. [115] in a 7 week supplementation study in pigs. Inulin-type fructans were added to the basal corn and soybean meal, which significantly decreased the expression of inflammation related genes, especially in lower gut mucosa. These different reports might be attributed to differences in the administered type of fructan (-mixtures) and other differences in experimental set up such as animal species or feeding protocol [116]. More and better designed studies in healthy experimental animals and humans are required to determine the specific immunomodulating effects of different inulin-type fructans. Although inulin-type fructan supplementation studies in healthy adult humans have been performed, immunological parameters were unfortunately often not measured. Immunological effects of inulin-type fructans have been studied in infants and elderly, but taking into account their immune status, these groups are to be categorized as immunocompromised, because the microbiota and

CHAPTER 1. IMMUNOLOGICAL PROPERTIES OF INULIN-TYPE FRUCTANS.

immune system of infants is not fully developed and the microbiota composition and immune function decreases qualitatively with age [117]. The only conclusions we can draw from the current supplementation studies in healthy human adults, studies in healthy experimental animals, as well as studies in infants and elderly, is that inulin-type fructan supplementation in healthy human adults will generally result in increased bifidobacteria numbers in the gut [75-79], increased levels of fecal sIgA [104-106], increased levels of IL-10 and IFN-γ in Peyer’s patches [104-107] and increased activity of different immune cells in the spleen [39,64,107,108,118], as these are the parameters which are most often reported to have been changed upon supplementation. The evidence for immunomodulation on a systemic level may be somewhat less strong than locally in the gut, however, the local cytokine levels in the gut may have more impact on an immune parameter such as prevention of infections, since the gut is the largest organ of the human body to be in such close contact with the outside world. In the following sections the effects of inulin-type fructans on immune structures are reviewed in the context of the different disease models which have been studied up to now.

1.7 Cancer and cancer animal models Two studies using experimental animal cancer models focused on immune parameters involved in anti-tumorigenic reactions. In a study by Roller et al. [119], the effects of probiotic Lactobacillus LGG and Bifidobacterium lactis Bb12, and FOS (“Raftilose,” chain length range 2-10, average 4, 100 g/kg of diet) synbiotic treatment on the immune system of rats were investigated in an azoxymethane (AOM)-induced colon cancer model. Synbiotic supplementation significantly restored Natural Killer cell-like cytotoxicity (p<0.01) and suppressed proliferative responsiveness of lymphocytes in Peyer’s patches of AOM-treated rats. It should be noted that no normal diet or placebo diet group was included in this study and that this alteration of responsiveness may be related to the background of the high fat diet. FOS supplementation significantly stimulated IL-10 production in Peyer’s patches and mesenteric lymph nodes of rats not treated with AOM (p<0.05). In pro- and synbiotic groups, IFN-γ production in Peyer’s patches was significantly decreased independent of AOM treatment (p<0.05). A study by Forest et al. [29] showed that short chain fructans (chain length range DP 1-4, mostly DP 3) reduced colon tumor incidence in intestinal neoplasia prone “adenomatous polyposis coli

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/multiple intestinal neoplasia” (Apc+/Min) mice via a functional local immune response. Apc is a tumor suppressor gene involved in development of colorectal cancer [120]. In the colons of Apc+/Min mice, FOS treatment restored large intestine-intraepithelial lymphocytes (LI- IELs) surface expression of anti-tumorigenic IL-15/IL-15Rα. In addition, FOS specifically induced a decrease in the proportion of CD4+ CD25+ LI-IELs which are considered to be tumor facilitating cells [29]. Publications on fiber supplementation in human cancer patients are rare, and mostly discuss FOS, inulin, or FOS-enriched inulin in synbiotic mixtures using Lactobacillus LGG and Bifidobacterium lactis Bb12 [30,121]; or Lactobacillus acidophilus La5, Lactobacillus bulgaricus, Bifidobacterium lactis Bb12, and Streptococcus thermophilus [121]. These studies demonstrated that supplementation induced secretion of IL-2 and IFN-γ by PBMCs and decreased bacterial translocation [30,31,121]. Results for immunological parameters in experimental animal cancer models and in human colon cancer patients upon inulin-type fructan supplementation are summarized in Table 2. Studies regarding tumor growth and outcome of disease upon supplementation with inulin-type fructans have demonstrated anti-carcinogenic properties in multiple experimental animal models [122-124] and cell lines [125,126]. As previously reviewed by Taper et al. [127] dietary treatment with inulin and/or FOS incorporated in the basal diet for experimental animals: (i) reduced the incidence of mammary tumors induced in Sprague-Dawley rats by methylnitrosourea; (ii) inhibited the growth of transplantable malignant tumors in mice; (iii) decreased the incidence of lung metastases of a malignant tumor implanted intramuscularly in mice. Moreover, dietary treatment with inulin and/or FOS (iv) significantly potentiated the effects of cytotoxic drugs and potentiated the effects of radiotherapy on solid form of transplantable lymphoid tumor. Especially the fermentation products of FOS-enriched inulin (“Synergy1”) i.e. SCFA and deoxycholic acid appear to limit tumor growth [128]. The most consistent findings were reductions in aberrant crypt foci, tumor incidence and metastasis in models which make use of chemically induced pre-neoplastic lesions or tumors in the colon of rats and mice [129-135]. Only one (preliminary) study in patients with colorectal adenomas was performed so far. This study was an open multicenter study on the effects of FOS. No beneficial effect was found on proliferation at the rectal crypts [136]. However, from experimental use of human ex vivo cells, significant anti-carcinogenic effects were reported [137-141]. Moreover, when applied in a synbiotic

CHAPTER 1. IMMUNOLOGICAL PROPERTIES OF INULIN-TYPE FRUCTANS.

CHAPTER 1. IMMUNOLOGICAL PROPERTIES OF INULIN-TYPE FRUCTANS. protocol, inulin has already shown beneficial effects on inhibition of carcinogenic processes [142]. The prescription of inulin-type fructans to colon/colorectal cancer patients should be applied with some caution, as there are reports that under certain circumstances, inulin-type fructans can actually enhance proliferation of adenomas [142-144]. It should be noted that these reports involve mice studies and results so far do not show these effects in human colon cancer. The mechanisms behind these effects are not clear and require further investigation. The production and balance of anti- inflammatory and pro-inflammatory/anti-tumorigenic cytokines such as IL- 10 and IL-12 respectively may play a role in these observations because anti-inflammatory cytokines could confer an inhibitory effect on the anti- tumorigenic properties of proinflammatory cytokines. On the other hand, inulin-type fructan supplementation has shown promising anti- tumorigenic properties [29,107] and further investigation into the underlying mechanisms of these processes, which may involve immunomodulation, is warranted.

1.8 Intestinal inflammation in patients and animal models Inflammatory Bowel Disease (IBD) is a group of inflammatory conditions of the GI tract. IBD is thought to be caused by a combination of genetic, environmental, and immunological factors [145]. The current paradigm is that these diseases result from a lack of tolerance to resident intestinal bacteria in genetically susceptible hosts [146-150]. The major types of IBD are Crohn’s Disease (CD) and Ulcerative Colitis (UC) [151]. CD and UC share similar symptoms but also differ in substantial features. CD can occur along the entire GI tract, whereas UC specifically affects the large intestine or colon. Another difference is that UC occurs more superficially in the gut lining while CD can also affect deeper layers of the intestine, and surrounding tissue. Another affliction of the intestine is Irritable Bowel Syndrome (IBS); a functional bowel disorder characterized by chronic abdominal pain, discomfort, bloating, and alteration of bowel habits in the absence of any detectable organic cause [152]. Evidence is slowly increasing that inulin-type fructan supplements exert beneficial effects on both bowel movements [80,153-155] as well as on GI immune parameters [33,156]. Supplementation studies have been performed in several animal colitis models as well as in patients with IBD or IBS (Table 3 and 4). Leenen and Dieleman [157] recently reviewed the effects of pre- and synbiotics on IBD/IBS. Few studies using prebiotics alone have been

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performed so far, however results look promising with regard to therapeutic use in treatment of IBD/IBS. FOS-enriched inulin supplementation lowered disease activity scores [33], fecal calprotectin (a gut inflammation marker) [34], and inulin supplementation lowered pouchitis disease index [158]. Sigmoidoscopy scores (i.e. inflammation scores of endoscopy of the distal colon) were reduced, and endoscopically and histologically verified reductions in inflammation of the mucosa of the ileal reservoir were observed [33,34]. mRNA levels of beta defensins 2, 3, and 4 (i.e. antimicrobial proteins) were significantly reduced by treatment while these markers are normally upregulated in active UC [159]. When applied in synbiotic set up, increased amounts of bifidobacteria in rectal mucosa were reported, and significant reductions in the expression of molecules that control inflammation in active UC [159]. TNFα, and IL-1a mRNA levels in mucosal tissue were significantly reduced (p=0.0175 and p=0.0379) but also, a significant increase in IL-10 positive CD11c+ DCs and expression of TLR2 and TLR4 was reported [33]. Beneficial effects in UC patients have been reported [158-160] as supplementation resulted in improvement of the full clinical appearance of chronic inflammation in patients receiving this therapy. In addition to reduction of intestinal inflammation, regeneration of epithelial tissue was observed. To our knowledge no trials have been conducted as yet to determine whether chronic supplementation with inulin-type fructans might ameliorate disease progression, prevent disease recurrence, or sustain periods of clinical remission. Currently available data was gathered in trials aimed at investigating the immediate effects [158-160], but it may be worthwhile in future studies to include longer trial periods. Regarding IBD, studies on treatment of chronic intestinal inflammation using inulin-type fructans have shown major benefits in experimental animal models of colitis [35,36,161-169] (Table 3). Several types of experimental animal models exist to mimic IBD; dextran sodium sulfate (DSS)-induced colitis [35,163,168,170], trinitrobenzene sulphonic acid (TNBS)-induced colitis [36,164,167,171], and an HLA-B27 transgenic colitis model [165,166] were studied in relation to inulin-type prebiotics. In most of these experimental animal models, supplementation rendered statistically significant beneficial effects by reduction of mucosal damage and reduced release of inflammatory mediators such as IL-1β [35,166,167], inducible nitric oxide synthase [167], myeloperoxidase activity [164,169,172] cyclooxygenase 2, and mucin 3 [167]. In conclusion, inulin-type fructans are promising agents to modulate the immune

CHAPTER 1. IMMUNOLOGICAL PROPERTIES OF INULIN-TYPE FRUCTANS. parameters involved in colitis. Underlying mechanisms of these effects are however still unclear and warrant more studies on the effects of inulin- type fructans in experimental animal colitis models. Moreover, in IBD patients, long term intervention studies and follow up are required to determine whether inulin-type fructans might have long term beneficial effects in treatment of these diseases. IBS is a common disorder of the GI tract and there is increasing evidence to support the role for immune activation in IBS [173-177]. In a number of patients the onset is triggered by acute gastroenteritis [178- 181]. Evidence of sustained immune activation has been found in these cases [182]. However, low-grade immune activation without previous acute gastroenteritis can also induce IBS symptoms [183,184]. The immune parameters most often increased in IBS, are IL-6 and IL-8 levels [185,186] and baseline and lipopolysaccharide (LPS) induced TNF-α, IL-1β and IL-6 levels in IBS patients PBMCs [187]. In experimental IBS rat models, increased TLR expression was found in the colonic mucosa of these animals [188,189]. These altered TLR responses may play a significant role in the enhanced immune activity in IBS [190]. The increased risk of developing IBS following gastroenteritis and the co- existence of a disturbed composition of the microbiota, elevated luminal gas production and immune activation, indicate that the gastrointestinal microbiota may be a therapeutic target in IBS. There are no recent clinical trials aimed at studying possible immunological benefits of inulin-type fructans in IBS, although previous prebiotic studies indicate potential health benefit at lower doses, i.e. an intake of 3.5-5g/day [191]. In the studies of Hunter et al., and Astegiano et al. [173,174], only FOS was applied so possible chain length effects have yet to be evaluated. Two other studies incorporated inulin-type fructans in a synbiotic mixtures (FOS “Actilight” and Bifidobacterium longum W11) / (IBS Active; inulin with Lactobacillus sporogenes, Lactobacillus acidophilus, Streptococcus thermophilus and other additives) [192,193]. In these synbiotic combinations with inulin, significant reduction in IBS pain symptoms, abdominal distension, and regulation of bowel movement occurred. Moreover, increased stool frequency, reduced abdominal pain and reduced bloating were reported. Olesen et al. [194] reported no beneficial effects in a study with IBS patients who were given chicory derived FOS. Concluding from these results, FOS is a promising agent in IBS therapies when combined with the appropriate probiotics and other cofactors. Future IBS studies including inulin-type fructan supplementation should

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include measurements of the immune parameters mentioned above to evaluate whether based on immunology, inulin-type fructans can provide therapeutic options.

1.9 Systemic immune benefits of inulin type fructan supplementation There are several clear physiological links between symptoms of rheumatoid arthritis (RA) and IBD, such as a shared inflammatory cytokine expression pattern and the success of several therapies in both diseases [163,191,195-197]. This may indicate that where inulin-type fructans might alleviate IBD symptoms, RA patients may equally benefit from such supplementation. Experimental data on the benefits of inulin-type fructans in RA are scarce but studies in an HLA-B27 rat models demonstrated reduced severity of colitis as well as reduced severity of arthritis [165,166]. After inulin-type fructan supplementation a significant reduction in inflammatory scores and pro-inflammatory cytokines was observed [166,191]. In adjuvant-induced arthritis in Wistar rats, and type II collagen-induced arthritis in DBA⁄1 J mice, α-GOS supplementation decreased erythema and swelling of limbs, and reduced the histopathological findings in the hind paw joints [198]. In conclusion, supplementation with inulin-type fructans and similar prebiotics such as α- GOS as mentioned above, seem to be a promising therapeutic strategy to reduce disease symptoms in experimental animal colitis models and may prove useful in human inflammatory diseases such as IBD, IBS and RA (Table 3 and 4) [165,166,197]. However, more experimental animal studies are first required to confirm these beneficial effects and their underlying physiological mechanisms [197].

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1.10 Allergy, infection, and immunization The effects of inulin-type fructans on allergies and immunization have been studied extensively in experimental animal models, mostly in combination with GOS administration, but protocols without GOS already show substantial immunological effects, which are summarized in Table 5. Many studies have been performed in pigs, where supplementation with inulin-type fructans mostly shows significant protective effects in infection models [199-203]. In one study by Milo et al. supplementation of piglets with 1% of inulin for 1 week did not affect immune parameters or infection symptoms upon inoculation with Salmonella typhimurium. In a study in dogs, inoculated with Salmonella typhimurium DT104, a 14 day supplementation with inulin or FOS (1%) improved food intake and enterocyte sloughing and attenuated fever [32]. Manhart et al. reported that a 16 day supplementation trial with 10% FOS induced an increased CD4+/CD8+ ratio in an experimental mouse model for LPS-induced endotoxemia [204]. Inulin-type fructans have been administered to infants and children because of their potential to modulate the intestinal microbiota and to benefit the development of an adequate innate and adaptive immune response (Table 6). In healthy infants, the most obvious effect upon supplementation was increased levels of IgA in fecal samples, which can protect against pathogens in the gut lumen [45,74,156,205,206]. Saavedra et al. [42] demonstrated an increase in blood IgG levels after measles vaccination in a 10 week supplementation study with OF/inulin (7/3, 0,2 g/kg BW/d) in 7-9 months old infants. However in a study by Duggan et al. [43] in which 6-12 month old infants were supplemented with OF (0.7g/d), no effect was observed on antibody response after vaccination with H. influenza type B vaccine. Results from these studies may be related to the specific pathogen, or the type of fructans used in the vaccine but further studies are required to investigate these differences. In both experimental animal studies and human studies, the use of inulin-type fructans has demonstrated beneficial effects on Th1 as well as on Th2 responses upon vaccination or sensitization protocols. Th1 cells normally drive the cellular immunity pathway to fight viruses and other intracellular pathogens, eliminate cancerous cells, and stimulate delayed- type hypersensitivity (DTH) skin reactions [48]. Th2 cells drive the humoral immunity pathway and up-regulate antibody production to fight extracellular organisms. In a study by Vos et al. [38] supplementation of mice which were vaccinated with influenza virus, a 9:1 mixture of

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GOS/FOS enhanced DTH responses dose-dependently, but a mixture of FOS/inulin did not. Fujitani et al. [49] describe anti-allergic effects of FOS in Nc/jic mice upon supplementation with 5% FOS. Schouten et al. [44] demonstrated that a mixture of GOS/FOS inhibited sensitization to orally supplemented whey in mice, but this was only effective when used in synbiotic combination with Bifidobacterium breve. Inulin-type fructans appear to modulate both reactions; stimulating the adaptive immune response in a Th1-direction upon vaccination or sensitization, inhibiting infections [38] or Th-2 related immune disorders such as allergies [37,44,49,207]. In these experimental animal studies, inulin-type fructan effectiveness was most pronounced when used in combination with either GOS or Bifidobacterium breve. On the other hand they can induce increased antibody production (IgA) as part of a Th2 response, increasing clearance of luminal pathogens and reducing the chance of pathogen tissue entry. Evidence for prevention of allergy incidence or atopic symptoms in infants was reported by Moro et al. [208], and Arslanoglu et al. [46,47] but in these studies inulin-type fructans were only supplemented in combination with GOS. In a study by Raes et al. [206] in which infants received breast milk, formula, or formula supplemented with GOS/FOS, no clear differences were observed in the investigated immune parameters, but a trend was observed that GOS/FOS supplementation tended to increase blood IgG levels. In conclusion, reduction of incidence of allergic symptoms or protective effects on development of allergy upon supplementation with inulin-type fructans have been shown in infants [45,74,156,205,206]. For elderly, promising results have been shown for supporting immune function, including protection against respiratory infections [209-213] (Table 6). Other groups of patients which may benefit from inulin-type fructan supplementation by means of Th-1/Th-2 modulation are pregnant women [214], or burn patients [215] but the small number of supplementation studies performed with these groups show no beneficial effects as yet. In a study in adult male smokers and non-smokers, 4 out of 23 immunological parameters were changed upon supplementation [216]. However, in the experimental set up of this study, inulin was incorporated in prebiotic bread, which also contained linseed and soy fiber, so observed effects cannot solely be attributed to inulin intake. More studies in healthy

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human subjects are required to assess the immunomodulatory potential of inulin-type fructans in healthy conditions.

1.11 Possible explanations for the inconsistencies Some of the inconsistencies in the studies focusing on the immunomodulating effects of inulin-type fructans are caused by pertinent differences in study design and the application of different types of inulin- type fructans. In many studies the type of fructan has not been clearly documented. In part this can be explained by the inconsistent use of nomenclature regarding chain length. The chain length should always be included as it has been shown that chain length is a determining factor for the beneficial effects [11,131,133,138]. The mechanisms behind this might be that specifically long chain fructans bind to typical receptors in the membrane and cluster them into membrane microdomains [99] or influence other membrane lipid dynamics [100-102] whereas the shorter chain fructans may lack these properties and exert their effects via different routes. These findings suggest that the chain length of fructans influences the efficacy in modulating immune functions and warrant further investigation [11]. Many of the studies mentioned in this literature overview report altered cytokine levels upon supplementation with inulin-type fructans. Nowadays a lot is known about the effects that cytokines or chemokines have on humans. The connection between inulin ingestion, the production of cytokines or chemokines, and the observed downstream health effects is gradually becoming clearer. On a chemical and cellular level inulin-type fructans can exert several effects and most pronounced effects are observed in the GALT. Several studies report modulation of IgG [39,105,111,138,206], and IgA [39,49,106,169,217] levels in serum and/or feces, changes in cytokine expression, mainly IFN-γ [39,105,107,119,121], IL-4 [64,218], IL-10 [105,107,119,218,219] and, IL-12 [39], and altered activity of spleen NK cells [110,119,140]. IFN-γ and IL-12 are factors responsible for Th1 differentiation, whereas IL-4 promotes Th2 subpopulations [48,62]. Regulatory T lymphocytes (Treg) are expanded in response to IL-10 and IL-10 skews the Th1/Th2 balance to Th2 in vivo by selectively blocking IL-12 synthesis by the antigen-presenting cells such as DCs [62,191]. In addition, IL-4 induces IgA synthesis by follicular B lymphocytes in the Peyer’s patches [61]. These B lymphocytes, plasma-cell precursors, will migrate, maturate, and undergo clonal expansion, and finally migrate to the intestinal lamina propria, where they finalize their

CHAPTER 1. IMMUNOLOGICAL PROPERTIES OF INULIN-TYPE FRUCTANS. maturation into IgA-secreting cells [63]. A great variety of factors can influence this migration, including the cytokines [65] induced by the inulin- type fructans. From this expression pattern of cytokines it appears that several types of T cell responses are induced by inulin-type fructans. Because of this complex interplay, and depending on the experimental set up, different outcomes may be observed.

1.12 Future studies with inulin-type fructans There are many biomarkers to quantify immunomodulation in human nutrition intervention studies, but the repercussions of variations in these markers are still unclear, especially in healthy subjects. A review by Albers et al. [25] discusses the suitability of a large panel of biomarkers for the evaluation of nutritional intervention. However, the choice of immune markers needs to be correlated with the particular condition that is being assessed, the relevant clinical end-points, and whether any immune markers are differentially expressed in disease and control populations [197]. Concluding from the current literature overview, recommended biomarkers, typically suitable for inulin-type fructan supplementation studies are IgG and IgA levels in serum and feces, cytokine expression patterns in the GALT, and NK cell activity in the spleen. Measurements of these markers may be affected by age and gender, and they might vary because of other external confounding factors such as stress, smoking, and alcohol intake [197]. This necessitates careful selection of the control subjects [197]. Patient populations which are likely to benefit from inulin- type fructan supplementation include allergic individuals, IBD patients, RA patients and likely also patients suffering from other chronic inflammatory afflictions. Well designed, randomized placebo controlled trials are useful to evaluate possible benefit these patient groups may derive from inulin- type prebiotic supplementation. The physiological effects of ingested inulin-type fructans are likely the combined effects of circumstances including an altered gut microbiota, the presence of produced fermentation products in the gut, and direct effects on gut epithelium and GALT, which complicates the analysis of the induced health effects. To investigate the direct effects, germ free experimental animal models or finely designed SPF experimental animal models and in vitro assays with suitable cell lines may shed more light on the induced physiological processes. Specific molecular or cellular effects which are reported repeatedly throughout literature are

CHAPTER 1. IMMUNOLOGICAL PROPERTIES OF INULIN-TYPE FRUCTANS.

important to consider in unraveling the mechanisms behind the observed health benefits and can form a basis for further explorative research. It appears that ingestion of inulin-type fructans affects a spectrum of immune reactions in the body including Th1 [30,39,119,121] Th2 [64,118], anti-inflammatory reactions [105,107,118,119,211], B cell activity [36,45,111,118,220] and NK cell activity [86,110,211]. Depending on the research question, in vitro studies may provide useful information about the typical processes which are induced upon ingestion. In vitro studies into the signaling capacity of inulin-type fructans could entail the use of NK cell activity reporter assays. Another signaling target could be B cell activation; Peyer’s patch DCs are able to induce B cell maturation and IgA production under influence of intestinal bacteria, via Peyer’s patch DC derived cytokines such as B-cell activating factor (BAFF) and A Proliferation-Inducing Ligand (APRIL) [221,222]. It is possible that inulin- type fructans exert the same effect. Another possibility of promoted IgA production under influence of inulin-type fructans could be due to the traditional activation of T cells in the Peyer’s patch follicles by DCs which have sampled the intestinal lumen and have encountered inulin molecules, followed by T helper cell-mediated B cell maturation and IgA production. Finally, it would be interesting to see whether direct ligation of B cell TLRs [221,223,224] by inulin-type fructans could result in IgA production. Regarding the altered cytokine levels in inulin-type fructan supplementation studies, it would be interesting to investigate which immune cell types are capable of recognizing inulin-type fructans and of mounting a subsequent cytokine response. This may be a wide range of cell types because many cell types express TLRs and CLRs. From there, results can be translated to the relevant populations which may actually come in contact with fructan molecules after their ingestion. Moreover, the capacity of APCs to report the presence of inulin-type fructans or parts of the molecules to effector cells is still an uncharted area of research which deserves further exploration.

1.13 Dietary fibers and intestinal barrier function By metadata analysis of dietary fiber studies in relation to health risks, it has now been generally established that dietary fiber intake is associated with a reduced incidence of disease [225-228] and disease-related mortality [229-232]. One of the suggested mechanisms by which disease is prevented is through the protection of the intestinal epithelial barrier [233]. By maintaining a proper barrier function, the luminal contents

CHAPTER 1. IMMUNOLOGICAL PROPERTIES OF INULIN-TYPE FRUCTANS. remain compartmentalized, and a state of tolerance is acquired. This physical barrier of epithelium is organized by focal contact points between the cells, known as tight junctions. Current literature reports demonstrate that many pathologies are in fact linked to an impaired intestinal barrier function [234]. This state of intestinal hyperpermeability, also known as leaky gut syndrome [235], leads to increased translocation of bacteria, endotoxins and other macromolecules, triggering aberrant local or peripheral immune reactions [236-239]. Intestinal hyperpermeability has been associated with the etiology and pathogenesis of atopic eczema [240,241], asthma [242,243], inflammatory bowel disease [244,245], diabetes [246-248], obesity [236,249], celiac disease [250-252], and diarrhea-predominant irritable bowel syndrome [253,254]. In addition, increased gut permeability is also implicated in several neurological disorders including schizophrenia [255], autism spectrum disorders [256], depression [256], and anxiety [256], suggesting that the compounds leaking through the intestinal wall can cause detrimental effects in organs as far removed and well-protected as the brain. Two prebiotic effects of dietary fibers are suggested to play a role in protection against these events, i.e. the stimulation of lactic acid producing bacteria, and the related increase in SCFA as fermentation products. For typical substrains such as L. rhamnosus GG [257,258], B. infantis [259], L. plantarum [98,260], and L. casei [261], it has already been shown that the presence of these bacteria in the gut optimizes the barrier function of the epithelium as indicated by improved tight junction protein content, reduction in permeability to chemicals, and reduced translocation of bacteria. Of SCFA, mainly butyrate [18,262,263], is capable of strengthening the epithelial barrier. Of the direct interaction of dietary fibers with epithelial cells and protection of their barrier function no information is available yet, to the best of our knowledge.

1.14 Chicory root pulp as valuable source of dietary fibers Besides a healthy lifestyle, sustainability is in this day and age a goal which both the food industry and consumers are interested in. After extraction of the inulin-type fructans from chicory, the root pulp is left over as byproduct. Usually this root pulp is applied as a livestock feed [264,265]. However, as the importance of a high fiber diet for a healthy lifestyle is becoming more evident, this byproduct could actually be a valuable

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source of dietary fibers for application in functional foods. To assess the potential effects of chicory root pulp, the studies described in this thesis were focused on creating a technology platform to test the beneficial properties and structure-response relationships of chicory root pulp fibers on the immune system and intestinal epithelial cells. Inulin-type fructans from chicory were used as model fibers to set up this platform to test fibers in primary cells and cell line models, and finally to test the application in human studies. Chicory root pulp (CRP) obtained after the extraction of inulin is rich in cell wall polysaccharides (CWP), and two major components of this chicory root pulp are cellulose [266] and pectin [267,268]. These two fiber types were analyzed for their immunomodulatory effects as well. In addition, the impact on barrier function of intestinal epithelial cells was tested. Finally, inulin-type fructans were evaluated for their in vivo immunomodulatory effects in a human supplementation study involving the analysis of hepatitis B vaccination efficacy combined with the analysis of peripheral blood lymphocyte subsets. This experimental set up is an integral part of the technology platform, and may serve as a backbone to study immunemodulation by dietary fibers in future human supplementation trials.

1.15 General aim of the studies in this thesis

As the importance of a healthy diet is gaining ground with the public, and since the field of nutritional immunology is expanding rapidly within the scientific world, studies into the immunological effects of dietary fibers are warranted, so that optimal use can be made of these naturally occurring plant products. With the identification of structure-response relationships, an additional advantage of these studies is that this knowledge can be used to make tailored functional food products with well-characterized effects. Based on the discussed immunological effects of dietary fibers we propose that the soluble and insoluble fibers from chicory root can be valuable nutritional compounds for improving and / or maintaining intestinal and systemic health by their beneficial effects on immune cells, as well as on the intestinal epithelial barrier function.

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DESIGN AND RATIONALE

As outlined in the preceding sections health effects of dietary fiber are well recognized but the mechanisms by which they accomplish health effects are not completely understood. Also the structure -response relationships for fibers are not well characterized so far. Inulin-type fructans were used as model fibers followed by analysis of the major chicory root pulp components cellulose and pectins. In Chapter 2 we tested whether inulin-type fructans can exert direct signaling when in contact with immune cells, and if the pattern recognition receptors TLRs and NODs are involved. Moreover, the specific chain length-dependency of the responses was characterized. In Chapter 3 we investigated whether inulin-type fructans exert promoting or protective effects on the barrier function of human intestinal epithelial cells. Barrier function was disrupted with the agent phorbol 12-myristate 13-acetate (PMA). The dynamics of this process were studied with regard to timing of fiber incubation, and fructan chain length effects. As inulin-type fructans have recently been identified as TLR ligands [269] (Chapter 2), and TLR2 is highly important in intestinal barrier regulation [270], the other aim of this study was to investigate whether receptor interactions with TLR2 on the epithelial surface are involved in inulin-type fructan mediated barrier modulation. In Chapter 4 the immunomodulatory capacity of the chicory root pulp component cellulose was tested. This was done using human peripheral blood mononuclear cells. The transcriptome of the cells was studied by gene chip array. Also barrier function measurements were performed with human intestinal epithelial cells using PMA as damaging agent. In Chapter 5 another major component of chicory root pulp, pectin, was studied for its immunogenic and barrier protective effects. In addition, the structure-response properties of pectins were studied using pectins with different degrees of methyl esterification. These pectins were tested for TLR activating properties, and for barrier function effects on human intestinal epithelial cells. In Chapter 6 we describe the results of a human supplementation study with different inulin-type fructan supplements. This study was performed with short chain-enriched inulin-type fructans, long chain-

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enriched inulin-type fructans, and fructose as placebo supplement. In this study, the effects of fructan supplementation on hepatitis B vaccination efficacy, as well as on peripheral blood lymphocyte subsets was analyzed. In conclusion, the results of this thesis are summarized and discussed in Chapter 7.

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235. Liu Z, Li N, Neu J. (2005) Tight junctions, leaky intestines, and pediatric diseases. Acta Paediatr 94: 386-393. 236. Fasano A. (2012) Leaky gut and autoimmune diseases. Clin Rev Allergy Immunol 42: 71-78. 237. Geurts L, Neyrinck AM, Delzenne NM, Knauf C, Cani PD. (2013) Gut microbiota controls adipose tissue expansion, gut barrier and glucose metabolism: Novel insights into molecular targets and interventions using prebiotics. Benef Microbes : 1-15. 238. Ruutu M, Thomas G, Steck R, Degli-Esposti MA, Zinkernagel MS, et al. (2012) Beta- glucan triggers spondylarthritis and crohn's disease-like ileitis in SKG mice. Arthritis Rheum 64: 2211-2222. 239. Seo YS, Shah VH. (2012) The role of gut-liver axis in the pathogenesis of liver cirrhosis and portal hypertension. Clin Mol Hepatol 18: 337-346. 240. Iemoli E, Trabattoni D, Parisotto S, Borgonovo L, Toscano M, et al. (2012) Probiotics reduce gut microbial translocation and improve adult atopic dermatitis. J Clin Gastroenterol 46 Suppl: S33-40. 241. Penders J, Stobberingh EE, van den Brandt PA, Thijs C. (2007) The role of the intestinal microbiota in the development of atopic disorders. Allergy 62: 1223-1236. 242. Benard A, Desreumeaux P, Huglo D, Hoorelbeke A, Tonnel AB, et al. (1996) Increased intestinal permeability in bronchial asthma. J Allergy Clin Immunol 97: 1173-1178. 243. Hijazi Z, Molla AM, Al-Habashi H, Muawad WM, Molla AM, et al. (2004) Intestinal permeability is increased in bronchial asthma. Arch Dis Child 89: 227-229. 244. Gassler N, Rohr C, Schneider A, Kartenbeck J, Bach A, et al. (2001) Inflammatory bowel disease is associated with changes of enterocytic junctions. Am J Physiol Gastrointest Liver Physiol 281: G216-28. 245. Soderholm JD, Peterson KH, Olaison G, Franzen LE, Westrom B, et al. (1999) Epithelial permeability to proteins in the noninflamed ileum of crohn's disease? Gastroenterology 117: 65-72. 246. Bosi E, Molteni L, Radaelli MG, Folini L, Fermo I, et al. (2006) Increased intestinal permeability precedes clinical onset of type 1 diabetes. Diabetologia 49: 2824-2827. 247. Sapone A, de Magistris L, Pietzak M, Clemente MG, Tripathi A, et al. (2006) Zonulin upregulation is associated with increased gut permeability in subjects with type 1 diabetes and their relatives. Diabetes 55: 1443-1449. 248. Vaarala O, Atkinson MA, Neu J. (2008) The "perfect storm" for type 1 diabetes: The complex interplay between intestinal microbiota, gut permeability, and mucosal immunity. Diabetes 57: 2555-2562. 249. Boroni Moreira AP, Fiche Salles Teixeira T, do C Gouveia Peluzio M, de Cassia Goncalves Alfenas R. (2012) Gut microbiota and the development of obesity. Nutr Hosp 27: 1408-1414. 250. Montalto M, Cuoco L, Ricci R, Maggiano N, Vecchio FM, et al. (2002) Immunohistochemical analysis of ZO-1 in the duodenal mucosa of patients with untreated and treated celiac disease. Digestion 65: 227-233. 251. Schulzke JD, Bentzel CJ, Schulzke I, Riecken EO, Fromm M. (1998) Epithelial tight junction structure in the jejunum of children with acute and treated celiac sprue. Pediatr Res 43: 435-441. 252. Vogelsang H, Wyatt J, Penner E, Lochs H. (1995) Screening for celiac disease in first- degree relatives of patients with celiac disease by lactulose/mannitol test. Am J Gastroenterol 90: 1838-1842. 253. Dunlop SP, Hebden J, Campbell E, Naesdal J, Olbe L, et al. (2006) Abnormal intestinal permeability in subgroups of diarrhea-predominant irritable bowel syndromes. Am J Gastroenterol 101: 1288-1294.

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254. Lee H, Park JH, Park DI, Kim HJ, Cho YK, et al. (2013) Mucosal mast cell count is associated with intestinal permeability in patients with diarrhea predominant irritable bowel syndrome. J Neurogastroenterol Motil 19: 244-250. 255. Severance EG, Yolken RH, Eaton WW. (2014) Autoimmune diseases, gastrointestinal disorders and the microbiome in schizophrenia: More than a gut feeling. Schizophr Res . 256. Lyte M, Cryan JF, editors. (2014) Microbial endocrinology: The microbiota-gut-brain axis in health and disease. New York: Springer New York. 257. Patel RM, Myers LS, Kurundkar AR, Maheshwari A, Nusrat A, et al. (2012) Probiotic bacteria induce maturation of intestinal claudin 3 expression and barrier function. Am J Pathol 180: 626-635. 258. Seth A, Yan F, Polk DB, Rao RK. (2008) Probiotics ameliorate the hydrogen peroxide- induced epithelial barrier disruption by a PKC- and MAP kinase-dependent mechanism. Am J Physiol Gastrointest Liver Physiol 294: G1060-9. 259. Ewaschuk JB, Diaz H, Meddings L, Diederichs B, Dmytrash A, et al. (2008) Secreted bioactive factors from bifidobacterium infantis enhance epithelial cell barrier function. Am J Physiol Gastrointest Liver Physiol 295: G1025-34. 260. Anderson RC, Cookson AL, McNabb WC, Park Z, McCann MJ, et al. (2010) Lactobacillus plantarum MB452 enhances the function of the intestinal barrier by increasing the expression levels of genes involved in tight junction formation. BMC Microbiol 10: 316-2180-10-316. 261. Castillo NA, de Moreno de LeBlanc A, M Galdeano C, Perdigon G. (2013) Comparative study of the protective capacity against salmonella infection between probiotic and nonprobiotic lactobacilli. J Appl Microbiol 114: 861-876. 262. Peng L, Li ZR, Green RS, Holzman IR, Lin J. (2009) Butyrate enhances the intestinal barrier by facilitating tight junction assembly via activation of AMP-activated protein kinase in caco-2 cell monolayers. J Nutr 139: 1619-1625. 263. Lewis K, Lutgendorff F, Phan V, Soderholm JD, Sherman PM, et al. (2010) Enhanced translocation of bacteria across metabolically stressed epithelia is reduced by butyrate. Inflamm Bowel Dis 16: 1138-1148. 264. Michel F, Thibault J, Barry J, de Baynast R. (1988) Preparation and characterisation of dietary fibre from sugar beet pulp. Journal of the Science of Food and Agriculture 42: 77-78-85. 265. Kelly P. (1983) Sugar beet pulp — A review. Anim Feed Sci Technol 8: 1-18. 266. Ramasamy US, Gruppen H, Schols HA. (2013) Structural and water-holding characteristics of untreated and ensiled chicory root pulp. J Agric Food Chem 61: 6077-6085. 267. Femenia A, Robertson JA, Waldron KW, Selvendran RR. (1998) Cauliflower (brassica oleracea L), globe artichoke (cynara scolymus) and chicory witloof (cichorium intybus) processing by-products as sources of dietary fibre. J Sci Food Agric 77: 511- 518. 268. Robert C, Emaga TH, Wathelet B, Paquot M. (2008) Effect of variety and harvest date on pectin extracted from chicory roots (cichorium intybus L.). Food Chem 108: 1008- 1018. 269. Vogt L, Ramasamy U, Meyer D, Pullens G, Venema K, et al. (2013) Immune modulation by different types of beta2-->1-fructans is toll-like receptor dependent. PLoS One 8: e68367. 270. Cario E. (2008) Barrier-protective function of intestinal epithelial toll-like receptor 2. Mucosal Immunol 1 Suppl 1: S62-6.

CHAPTER 2

Immune Modulation by different types of β2→1-FRUCTANS IS Toll-like receptor dependent

Leonie Vogt1, Uttara Ramasamy2, Diederick Meyer3, Gerdie Pullens4, Koen Venema5, Marijke M. Faas1,6, Henk A. Schols2, Paul de Vos1

1 Department of Pathology and Medical Biology, Division Medical Biology, Groningen University, University Medical Center Groningen 2 Laboratory of Food Chemistry, Wageningen University, PO Box 8129, 6700 EV Wageningen 3Sensus B.V., Borchwerf 3, 4704 RG Roosendaal 4Cosun Food Technology Centre, Oostelijke Havendijk 15, 4704 RA, Roosendaal 5TNO Quality of Life, Department of Biosciences, PO Box 360, 3700 AJ Zeist 6Department of Obstetrics and Gynaecology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, the Netherlands

Published in PLOS ONE, vol. 8, p. e68367, Jul. 2013.

CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT.

Abstract

Introduction: β2→1-fructans are dietary fibers. Main objectives of this study were 1) to demonstrate direct signaling of β2→1-fructans on immune cells, 2) to study whether this is mediated by the pattern recognition receptors Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain- containing proteins (NODs), and 3) to relate the observed effects to the chain length differences in β2→1-fructans. Methods: Four different β2→1-fructan formulations were characterized for their chain length profile. Human peripheral blood mononuclear cells (PBMCs) were stimulated in vitro with β2→1-fructans, and production of IL-1Ra, IL-1β, IL-6, IL-10, IL-12p70, and TNF-α was analyzed. Reporter cells for TLRs and NODs were incubated with β2→1- fructans and analyzed for NF-κB/AP-1 activation. Results: Cytokine production in human PBMCs was dose-, and chain length-dependent. Strikingly, short chain enriched β2→1-fructans induced a regulatory cytokine balance compared to long chain enriched β2→1-fructans as measured by IL-10/IL-12 ratios. Activation of reporter cells showed that signaling was highly dependent on TLRs and their adapter, myeloid differentiation primary response protein 88 (MyD88). In human embryonic kidney reporter cells, TLR2 was prominently activated, while TLR4, 5, 7, 8, and NOD2 were mildly activated. Conclusions: β2→1-fructans possess direct signaling capacity on human immune cells. By activating primarily TLR2, and to a lesser extent TLR4, 5, 7, 8, and NOD2, β2→1-fructan stimulation results in NF-κB/AP-1 activation. Chain length of β2→1-fructans is important for the induced activation pattern and IL-10/IL-12 ratios.

CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT.

Introduction

2.1 Background High fiber intake is associated with lower mortality in subjects suffering from circulatory, digestive, and non-cardiovascular non-cancerous inflammatory diseases [1-3]. These associations are similar for men and women and are observed in most countries even after careful adjustment for potentially confounding lifestyle and dietary differences [4]. The mechanisms by which fibers contribute to reduced mortality remain to be identified but have been suggested to be of both metabolic and immunological nature [5]. Cereal fibers have been associated with lower concentrations of inflammatory biomarkers [4], but there is an urgent need for studies addressing specific fibers [4] to obtain a better understanding of mechanisms and components underlying protective associations. One of the many types of dietary fibers which have been reported to elicit health benefits are linear β2→1-fructans (also named inulin-type fructans, ITFs, or fructooligosaccharides). These fructans are made up of fructose subunits, can vary considerably in chain length, and occur with or without the presence of a terminal glucose moiety [6]. The fructose oligomers are denoted as Fn or GFn (n = number of fructose subunits, GFn = fructose chain terminated with a glucose molecule) [6]. The chain length can also be denoted by the Degree of Polymerization (DP), representing the number of monosaccharide subunits in the chain [6]. Whether these variations in composition cause different physiological responses is currently unknown. Ingestion of β2→1-fructans induces specific effects on the immune system, as recently reviewed [7-11]. Changes in immunological parameters have been reported in the gut lumen, Peyer’s patches, spleen, and blood [12-14]. These effects could be induced via a prebiotic mechanism by stimulating beneficial (“probiotic”) bacteria in the intestine, such as bifidobacteria and lactobacilli [10]. This can coincide with modulation of fermentation products such as increased production of short chain fatty acids [15,16], which affect the recruitment of lymphocytes to inflammatory sites [17,18], and suppress the production of pro-inflammatory cytokines and chemokines [18-20]. Besides through these more indirect effects on the microbiota and its fermentation products, another pathway of modulation is gaining attention, i.e. through direct signaling on immune cells. This theory is supported by new insights into the structural and functional makeup of the intestine [21,22]. Some dietary fibers such as β-glucans, modulate the human immune system by

CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT. binding to pattern recognition receptors (PRRs) on cells of the innate immune system, such as Dectin-1 and Toll-like receptors (TLRs) [23-25]. These PRRs recognize so-called pathogen-associated molecular patterns (PAMPs), which are small molecular motifs found on groups of pathogens but also on many microorganisms with beneficial or no effects on host health [26]. Because of their ability to recognize specific carbohydrate moieties and elicit immune responses, we hypothesized that PRRs, and more specifically TLRs and NODs are activated by β2→1-fructans, and that this is one of the pathways by which β2→1-fructans can influence the human immune system [27,28]. As little knowledge is available on the specific immunomodulating effects of different types of β2→1-fructans, we investigated the effect of β2→1-fructans with different chain lengths on cytokine release by human Peripheral Blood Mononuclear Cells (PBMCs) and we studied whether and which PRRs are involved in β2→1-fructan recognition. Our studies support the concept that ITFs can signal directly on immune cells via TLRs and that chain length differences in ITFs can differentially affect immunological parameters.

methods

2.2 Ethics Statement Chemical analyses (HPAEC and HPSEC) were performed at Wageningen University. Other experiments, including blood sampling of human volunteers, were conducted within the University Medical Center Groningen, in the Netherlands. Written informed consent was obtained, and data were analyzed and presented anonymously. This research and consent procedure have been approved by the ethical review board of the University Medical Center, Medisch Ethische Toetsingscommissie University Medical Center Groningen, as documented in the approved application ‘‘2007/255’’. All clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.

2.3 High Performance Anion Exchange Chromatography Four different β2→1-fructan formulations were selected and their specific chain length profiles (range and distribution) were characterized by High Performance Anion Exchange Chromatography (HPAEC). The applied β2→1-fructans were extracted from chicory root and comprise a mixture of different chain lengths varying from DP 2 to 60, referred to as inulin or

CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT. native inulin [29]. These chicory root β2→1-fructan chains are terminated by a glucose molecule [6]. Fructans of varying DP can be acquired by partial enzymatic hydrolysis of this extract resulting in GFn and Fn chains with GFn ranging from DP 2 to 10 [6]. Another way to obtain β2→1- fructans is to synthesize them from sucrose, resulting in GFn type fructans with DP 2-4 [30]. The following β2→1-fructan formulations were analyzed: ITF I (Frutalose©OFP) ITF II (Frutafit©CLR), ITF III (Frutafit©IQ), and ITF IV (Frutafit©TEX!). β2→1-fructan formulations were provided by Sensus BV, Roosendaal, The Netherlands. Endotoxin levels (endotoxin units, EU) of all used β2→1-fructan samples were assessed by Toxikon (Leuven, Belgium) and were below 0.3 x 10-3 µg-1. For HPAEC, a Dionex ICS 3000 system (Dionex), equipped with a Dionex CarboPac PA-1 column (2 x 250 mm) in combination with a CarboPac PA-1 guard column (2 x 50 mm) was used. Sample concentrations of 0.05 – 0.1 mg/ml and partial-loop injection of 10 µl were applied. The system was equipped with pulsed amperometric detection. Elution was performed at 0.3 ml/min. and oligomers were separated using a gradient as follows: 0-400mM NaOAc in 100 mM NaOH within 40 min., followed by a 5 min. washing step (1M NaOAc in 100 mM NaOH), and 15 min. equilibration (100 mM NaOH).

2.4 High Pressure Size Exclusion Chromatography β2→1-fructan formulations were tested for their elution profile using High Performance Size Exclusion Chromatography (HPSEC) on Dionex Ultimate 3000 HPLC (Dionex, Sunnyvale, CA, USA). The analysis was performed on three TSK-Gel columns connected in series (4000-3000-2500 SuperAW; 150 x 6 mm). The columns were preceded by a TSK Super AW-L guard column (35 x 4.6 mm). All columns were from Tosoh Bioscience (Tokyo, Japan). Elution was performed at a flow rate of 0.6 ml/min. using sodium nitrate (0.2 M) as the eluent. A volume of 20 µl of the sample (2.5 mg/ml) in millipore water was injected and eluted at 55°C. Solubles were detected using a refractive index detector, Shodex type RI 101 (Showa Denko, Japan). The software used for acquiring the data was Chromeleon version 7. Molecular weight distribution of polysaccharides was determined using pullulan standards (Polymer Laboratories, Varian Inc., Palo Alto, CA, USA) in the molecular mass range of 0.18 - 790 kDa.

CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT.

2.5 Isolation Human Peripheral Blood Mononuclear Cells Human PBMCs were used to study whether β2→1-fructans can signal on immune cells, especially since PBMCs express many PRRs such as TLRs [31,32] and NODs [33]. Peripheral blood from human volunteers was collected in heparinized tubes (15 IU/ml lithium-heparin, Becton Dickinson B.V., Breda, The Netherlands) and PBMCs were isolated by Ficoll density gradient separation (Lymphoprep, Axis-Shield, Oslo, Norway). Cells were kept in RPMI1640 medium (Gibco, Life Technologies, Bleiswijk, The Netherlands) supplemented with 10% Fetal Bovine Serum (FBS, HyClone, Thermo Scientific, Breda, The Netherlands) and 50 µg/ml gentamicin (Gibco).

2.6 Cytokine expression As a read out for PBMC activation, cytokine production in the medium was analyzed after stimulation of PBMCs with a concentration series of β2→1- fructans. To evaluate whether β2→1-fructans induce a more anti- inflammatory or pro-inflammatory effect, and whether chain length profile would affect this balance, the IL-10/IL-12 ratio was calculated for each β2→1-fructan formulation at 1 µg/ml or 100 µg/ml. In this context, a higher IL-10/IL-12 ratio is representative of a more anti-inflammatory effect [34,35]. Human PBMCs (n=6 ; 3 males, 3 females, age 25-42 yr., healthy non-smoker volunteers) were seeded in a 24 wells plate at a density of 2 x 106 cells/well with a final volume of 1 ml/well. To characterize possible effects of different chain length profiles on cytokine expression, the β2→1-fructan formulations ITF I to IV were dissolved in culture medium and added to the PBMCs at final concentrations ranging from 1 to 100 μg/ml. After 24h of incubation (37°C, 5% CO2) cytokine levels in the supernatant were measured using a Bio-PlexTM premixed cytokine assay, human 6-plex group I; cat.#: M5000B6CPS, control 5022016, according to the manufacturer’s instructions. (Bio-Rad Laboratories, Veenendaal, The Netherlands). This customized kit simultaneously measured human IL-1Ra, IL-1β, IL-6, IL-10, IL-12p70, and TNF-α. Concentration series of cytokine standards were prepared for the appropriate concentration range, and coupled beads were diluted ten times, resuspended, and added to a pre-wetted filter plate. After washing the plate twice, standards, negative controls and samples (all in duplicate) were transferred into the plate (50 µl per well), and the plate was sealed and incubated on a shaker at room temperature (RT) for 30 min. After incubation, the plate was washed three times, detection antibodies were resuspended and diluted ten times and 25 µl was added to each well. The

CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT. plate was incubated on a shaker at RT for 30 min., and after washing three times, 50 µl of streptavidin-phycoerythrin was added to each well and the plate was incubated on a shaker at RT for 10 min. After washing the plate three times, 125 µl of assay buffer was added per well, the plate was incubated on a shaker for 5 min. and fluorescence was measured using a Luminex 100 System and StarStation software. All procedures as of the incubation with detection antibodies were performed in the dark.

2.7 Cell culture of reporter cell lines Selection media, Normocin antibiotic, Quanti-blue reagent, TLR agonists and the following TLR-, and NOD-reporter cell lines were acquired from InvivoGen (InvivoGen, Toulouse, France). Two THP-1 human acute monocytic leukemia reporter cell lines were acquired from InvivoGen, both endogenously expressing human TLRs and with inserted construct for Secreted Embryonic Alkaline Phosphatase (SEAP) coupled to the NF- κB/AP-1 promoter. The first of these THP-1 cell lines carries extra inserts for MD2 and CD14 to boost TLR signaling, and the second THP-1 cell line expresses only a truncated, non-functional form of the TLR adapter MyD88. Nine different human Embryonic Kidney (HEK293)-Blue reporter cell lines were purchased from InvivoGen, each with a different inserted construct for either human TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, NOD1, or NOD2, and all nine cell lines carrying an inserted construct for Secreted Embryonic Alkaline Phosphatase (SEAP) coupled to the NF- κB/AP-1 promoter. Both THP-1 cell lines were maintained in RPMI1640 containing 10% heat inactivated FBS, NaHCO3 (Boom B.V. Meppel, The Netherlands; 1,5 g/l), L-glutamine (2 mM), glucose (4,5 g/l), HEPES (10 mM), sodium pyruvate (1 mM) penicillin/streptomycin (50 U/ml and 50 µg/ml), and Normocin (100 µg/ml), all from Sigma-Aldrich Chemie B.V., Zwijndrecht, The Netherlands. Both THP-1 cell lines were kept at a concentration of 5x105 cells/ml. HEK-Blue cells were maintained in DMEM (Life Technologies Europe B.V.) containing 10% heat inactivated FBS, L- glutamine (2 mM), glucose 4,5 g/l), penicillin/streptomycin (50 U/ml and 50 µg/ml), and Normocin (100 µg/ml). HEK cells were grown to ≈ 80% confluency. After culturing for 3 passages, all reporter cell lines were maintained in selection media according to the manufacturer’s protocol.

2.8 Reporter cell stimulations and Quanti-BlueTM analysis THP-1 cells were centrifuged for 5 min. at 300 g to collect the cells and resuspended to the cell density specified by the manufacturer’s protocol (Table 1). In a flat bottom 96 wells plate, 100 µl of this cell suspension per

CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT.

well was stimulated for 24h (37⁰C, 5% CO2) with 10 µl of stimulus, i.e. concentration series (1 µg/ml - 2 mg/ml) of β2→1-fructan formulations ITF I to IV, or the relevant positive control as indicated by the manufacturer’s protocol (Table 1). Plain culture medium and endotoxin-free water were applied as negative controls. After incubation, 20 µl of cell culture medium of stimulated reporter cells was incubated with 180 µl of Quanti-blue reagent in a new flat bottom 96 wells plate for 45 min at 37⁰C and SEAP activity (absorbance), representing activation of NF-κB/AP-1, was measured at 650 nm on a VersaMax microplate reader (Molecular Devices GmbH, Biberach an der Riss, Germany) using SoftMax Pro Data Acquisition & Analysis Software. HEK-Blue reporter cells were rinsed with medium to detach them from the culture flask and cells were resuspended to the cell density specified by the manufacturer’s protocol (Table 1). 180 µl of cell suspension per well was stimulated for 24h (37˚C, 5% CO2) with 20 µl of β2→1-fructan, endotoxin free water, plain culture medium or positive control (Table 1) in a flat bottom 96 wells plate. For all HEK cell lines, after incubation, analysis of SEAP was performed in the same way as described for the THP-1 cells.

Table 1. Cell densities and positive controls used in reporter cell stimulations

2.9 Statistical analysis Significance levels were determined by parametric Student’s t-test for unpaired observations (two-tailed) or by non-parametric Mann-Whitney U-test for unpaired observations (two-tailed). Results are expressed as mean  SEM or mean  SD respectively. A P-value < 0.05 was considered statistically significant. P-values < 0.05 are denoted with *, P-values < 0.01 are denoted with **, and P-values < 0.001 are denoted with ***.

CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT.

Results

2.10 Chemical characterization of β2→1-fructan formulations The oligomer profiles of four β2→1-fructan formulations were characterized using HPAEC and HPSEC. Figure 1A depicts the oligosaccharide range and relative response per oligomer of the β2→1- fructan formulations tested with HPAEC. Figure 1B represents the HPSEC analysis of the four different β2→1-fructan formulations, showing the elution patterns as a measure for the molecular size distributions of these compounds in kDa. The HPSEC analysis corroborated the observed chain length profiles from the HPAEC analysis and in addition this method rendered a visual representation of the degree in which the different chain lengths are present per analyzed formulation. The most important differences can be observed between ITF I and II as compared to ITF III and IV. ITF I can described as fructooligosaccharide (FOS) with mostly chain lengths of <10, and ITF II can be described as a FOS-enriched inulin, with a large proportion of chains smaller than DP10, but also containing chains with DP up to 25. ITF III and IV are described as “inulin” due to their broad range of chain lengths present (up to DP60). ITF I and II contain mostly chains of the type

GF3, GF4, and GF5, (i.e. starting with a glucose molecule followed by 3, 4, or

5 fructose subunits) and F3, F4, or F5 (chains consisting only of 3, 4, or 5 fructose moieties). Between ITF I and II, ITF I contains relatively more Fn type oligosaccharides and ITF II contains relatively more GFn type molecules. ITF III and IV consist solely of the GFn type fructans while ITF I and II consist of both GFn and Fn fructans. Glucose and fructose monomers, and GF and GF2 (i.e. dimers of glucose and fructose subunits and trimers made up of one glucose subunit and two fructose subunits) are present in all four ITFs.

2.11 β2→1-fructans induce cytokine production in human PBMCs To gain insight into dose effects and to determine which cytokines are induced by β2→1-fructans we first screened for a panel of pro- and anti- inflammatory cytokines, i.e. IL-1β, IL-6, IL-12, TNF-α, IL-1Ra, and IL-10, after stimulating human PBMCs for 24 hours with β2→1-fructans. This was done for ITF I, in doses of 1/ 2,5/ 5/ 10, and 100 μg/ml. As shown in Figure 2, IL-1Ra expression was decreased for lower doses (2,5 to 10 µg/ml β2→1-fructans) and increased at 100 µg/ml β2→1-fructans (p<0.05, panel A).

CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT.

Figure 1. HPAEC profiles and HPSEC elution patterns of different inulin-type fructan formulations. Figure A depicts the fructose (F) and glucose (G) monomers, dimers, and fructan oligomers present in the fructan formulations. GFn chains are terminated by a glucose molecule, and Fn chains consist of only fructose moieties. In both cases, n represents the number of fructose moieties in the chain. Figure B depicts the elution patterns as a measure for molecular weight distribution profiles of the four different inulin- type fructan formulations in kDa.

CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT.

Figure 2. Induction of cytokines by ITF I, in a dose range of 0 to 100 mg/ml. Statistical significance levels were determined with a parametric Student’s t-test for unpaired observations (two-tailed). Mean and SEM of cytokine production is plotted as percentage of controls, which were set to 100% (n = 4). Panels A to F show the results for IL-1Ra, IL-1b, IL- 6, IL-10, IL-12, and TNF-a respectively.

The same pattern was observed for IL-6; expression was decreased for 2.5 µg/ml (p<0.05) and a substantial increase was observed at 100 µg/ml (p<0.01, panel C). IL-10 expression was increased, especially at 1 µg/ml (p<0.001) and 100 µg/ml (p<0.0001, panel D). A small increase for TNFα was observed at 100 µg/ml (12%, p<0.05, panel F). Since a dose as low as

CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT.

1 µg/ml induced a significant increase for IL-10, and 100 µg/ml induced significant and substantial increases, these low end and high end doses were subsequently used for further cytokine measurements in PBMCs.

2.12 Inulin-type fructan-induced cytokine production by human PBMCs is chain length dependent To gain insight into size-response relationships, we investigated whether the chain length of β2→1-fructans has an effect on the type and quantity of cytokines produced by PBMCs. ITF I to IV were tested at the concentrations of 1 and 100 μg/ml. IL-6 and IL-10 were mainly induced by ITF I and ITF II (Figure 3) which can therefore be mainly attributed to short chain molecules enriched in these formulations, i.e. F2-F5 and GF2-GF5 respectively. Production of IL-12 was slightly increased by ITF II and strongly induced by ITF III and IV, indicating that longer chains (>DP8, GFn type) have to be held responsible for this effect.

Figure 3. Induction of cytokines by inulin-type fructan formulations with different chain length (DP range), at 0, 1, and 100 mg/ml. Statistical significance levels were determined with a non-parametric Mann-Whitney U-test for unpaired observations (two-tailed). Mean and SD of cytokine expression are plotted as percentage of controls (represented by 0 mg/ml), which were set to 100% (n = 6). Panel A to D represent cytokine expression induced by ITF I to IV respectively.

CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT.

Similarly, the highest production of TNF-α was observed after incubation with ITF IV. These combined results showed that chain length profile of β2→1-fructan formulations is an important determinant of the cytokine profile which is induced upon stimulation. Next, the IL-10/IL-12 ratios were calculated to quantify whether there is a correlation between chain length profile and cytokine balance (Figure 4). IL-10/IL12 ratios can be applied to calculate the regulatory effects of bioactive food components [36]. Controls were set to 100, and a ratio of more than 100 was considered to be regulatory or anti- inflammatory, whereas a ratio lower than 100 was considered to be proinflammatory. Strikingly, the IL-10/IL-12 ratios gradually decreased in the sequence of ITF I > ITF II > ITF III > ITF IV, indicating that shorter chain fructans induce a regulatory cytokine balance in human PBMCs compared to longer chain fructans. When taking the structural differences into consideration (Figure 1) it can be concluded that the molecules F2-F5 and/or GF2-GF5, which are enriched in ITF I and II, skew the IL-10/IL-12 ratio in PBMCs more towards IL-10, and thus induce a more anti- inflammatory balance.

Figure 4. Ratio of IL-10/IL-12 upon incubation of PBMCs with different chain length inulin- type fructans. PBMCs (n = 6) were stimulated with 1 mg/ml (panel A) and 100 mg/ml (panel B) inulin-type fructans for 24h. Statistical significance levels were determined with a non- parametric Mann-Whitney U-test for unpaired observations (two-tailed). Mean and SD of the IL-10/IL-12 ratios is plotted for the different inulin-type fructans as percentage of controls, which were set to 100% (n = 6) and horizontal bars indicate the significant differences between inulin-type fructan treatments.

CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT.

2.13 TLR mediated activation of NF-κB/AP-1 by β2→1-fructans is dependent on the presence of functional TLR adapter MyD88 Next, we investigated whether the activation of immune cells can be explained by activation of PRRs by the β2→1-fructans. To this end we applied the following strategy.

Figure 5. NF-kB/AP-1 activation in THP-1 MD2-CD14 and THP-1 defMyD reporter cells. Statistical significance levels were determined with a non-parametric Mann-Whitney U-test for unpaired observations (two-tailed). Mean and SD of NF-kB/AP-1 activation by inulin- type fructans of different chain lengths (ITF I, II, III and IV) are plotted as percentage of negative controls (unstimulated cells), which were set to 100% for both cell lines. LPS stimulation was used as a common positive control for TLR4/MyD88 signalling to NF- kB/AP-1 in the functional THP-1 MD2-CD14 cell line. TriDAP was used as a positive control in THP-1 DefMyD cells, which induces MyD88-independent signalling to NF-kB/AP-1. Panel A to D represent cytokine expression induced by ITF I to IV respectively. Only concentrations which induced activation in the THP-1 MD1-CD14 cells are shown for comparison of these concentrations in the THP-1 DefMyD cells.

CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT.

First, β2→1-fructans were tested for the ability to induce NF-κB/AP-1 transcription in a THP-1 reporter cell line which endogenously expresses all TLRs, and carries extra inserts for co-signaling molecules CD14 and MD2 to increase the TLR-mediated responses. NF-κB and AP-1 are essential transcription factors in signaling for cytokine release [37]. ITF I and II induced a statistically significant elevation of NF-κB/AP- 1 activation in THP-1 MD2-CD14 cells, while ITF III and ITF IV only induced activation at higher doses (Figure 5). The strongest activation was observed for ITF II, which is mainly short chain GFn. Next, we investigated whether the activation was TLR-dependent by testing the β2→1-fructans on a specific THP-1 reporter cell line carrying the truncated TLR adapter molecule MyD88. These THP-1 DefMyD cells express only non-functional MyD88, whereas the functional MyD88 is an essential adapter molecule for TLR2, 4, 5, 7, 8, and 9 signaling. The β2→1-fructan doses which significantly activated THP-1 MD2-CD14 cells were subsequently tested in the THP-1 DefMyD cells. β2→1-fructan mediated NF-κB/AP-1 activation appeared to be TLR dependent as the activation pattern as observed in THP-1 MD2-CD14, was virtually absent in the MyD88 deficient cell line. This indicated that β2→1-fructan mediated signaling to NF-kB/AP-1 was TLR2, 4, 5, 7, 8, and/or 9 dependent. Slight activation of THP-1 DefMyD cells by ITF IV suggests that other activation pathways independent of MyD88 may also have been induced by high concentrations of the longer chain β2→1-fructans which are enriched in this ITF.

2.14 β2→1-fructans induce strong dose dependent activation of TLR2, slight activation of TLR4, 5, 7, 8, and NOD2, but no activation of TLR3, TLR9, or NOD1. The results from the THP-1 cell stimulations showed that TLRs are involved in β2→1-fructan signaling. Next, we investigated which TLRs are specifically activated by β2→1-fructans and whether the intracellular PRRs NODs were activated. To this end, HEK reporter cell lines, each carrying one construct for a specific TLR or NOD, were stimulated with β2→1- fructans in the same way as described for the THP-1 cells (Figure 6). Strikingly, HEK cells with TLR2 construct were strongly and dose dependently activated by the β2→1-fructans. Moreover, this response was chain length dependent, as NF-κB/AP-1 activation in HEK TLR2 cells increased with increasing chain length in the sequence of ITF I < ITF II < ITF III < ITF IV. In these cells, long chain fructans induced the strongest activation, up to 6-fold induction as compared to control. To a lesser extent, HEK cells carrying either TLR4, 5, 7, 8, or NOD2 were activated

CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT. upon β2→1-fructan stimulation. HEK cells expressing TLR3, 9 or NOD1 were not significantly activated by β2→1-fructan stimulation (data not shown). These data combined show that TLR binding was β2→1-fructan type-, and thus chain length dependent.

Discussion

We hypothesized that β2→1-fructans can affect the immune status through physical contact with pattern recognition receptors (PRRs) on gut immune cells such as IELs [38] or DCs [27,28]. As TLR activation in PBMCs leads to cytokine production [31,39], we used these cells to test whether β2→1-fructans would also induce production of cytokines. Both anti- inflammatory and proinflammatory cytokines were produced, demonstrating that β2→1-fructans have the ability to activate human immune cells. Interestingly, chain length proved to be an important factor in skewing the cytokine balance. Anti-inflammatory IL-10 was strongly induced by short chain β2→1-fructans and a striking correlation was observed between the ratio of IL-10/IL-12 and the chain lengths of the fibers. The IL-10/IL-12 ratio has previously been used to evaluate the effects of probiotic bacteria on immune responses [40,41], and to describe anti-inflammatory effects of butyrate [42]. It is therefore useful to apply the same approach to study the effect of prebiotic fibers. It can be concluded on the basis of our data that the short chain molecules which are more abundant in ITF I and II, skew the IL-10/IL-12 ratio in PBMCs more towards IL-10, and thus induce a more anti- inflammatory balance. When considering that the proportion of short chain fructans of the GFn type are also distributed as ITF I > ITF II > ITF III, the results also suggest that specifically the short chain fructans of the GFn type determine the outcome of this cytokine ratio. In spite of the fact that longer chains induced more IL-12 compared to shorter chains, the induced IL-10/IL-12 ratio is not significantly different from the ratio measured in the control cells, indicating that they are more pro-inflammatory as compared to the shorter chains but not pro-inflammatory per se, when compared to controls. IL-6 production was observed for higher concentrations of ITF I and II. Although IL-6 production is generally regarded as proinflammatory, IL-6 can exert several anti-inflammatory effects such as inhibition of TNF-α function [43], and activation of IL-1Ra and IL-10 [44].

CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT.

Figure 6. NF-kB/AP-1 activation of HEK cell lines overexpressing separate TLRs or NODs. Statistical significance levels were determined with a non-parametric Mann-Whitney U-test for unpaired observations (two-tailed). Mean and SD of NF-kB/AP-1 activation in HEK cell lines stimulated with inulin-type fructans for 24 h are plotted as percentage of unstimulated controls, which were set to 100%. Dosages are plotted in mg/ml. Endotoxin free H2O was used as an additional negative control and per cell line the relevant positive controls were applied as mentioned in Table 1.

Only few studies have addressed in vitro stimulation of immune cells by β2→1-fructans and cytokine profiles. In a study of Eiwegger et al. [45] three types of prebiotic oligosaccharides, including a mixture of short chain galactooligosaccharides (scGOS) and long chain β2→1-fructans were analyzed for the induction of a selected panel of cytokines in cord blood mononuclear cells (CBMCs). The mixture containing long chain β2→1-

CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT. fructans did not induce IL-10 in these experiments which corroborates our findings. Long chain β2→1-fructans alone, or short chain β2→1-fructans were not tested in the Eiwegger study, which would allow for a more complete comparison with our results. In supplementation studies in experimental animals, the observed immune effects with β2→1-fructans such as increased cytokine productions, increased serum and secretory immunoglobulins [13,46-49], and increased numbers of IL-10+, TLR2+, and TLR4+ DCs [50] could be due to direct effects of β2→1-fructan-mediated TLR activation in the intestine, be it on immune cells or intestinal epithelial cells [51]. However, the underlying mechanisms in these in vivo experiments are likely to be more complex and are probably the result of combined direct and indirect effects in the intestine. Also other studies corroborate our findings on chain length effects and differences in induced immune parameters. In a study where rats were supplemented with shorter chain β2→1-fructans (DP2-8), increased ex vivo secretions of IL-10 were observed in cells from the Peyer’s patches [13,52-54] and mesenteric lymph nodes [53]. In another study by Ito et al. the effect of different chain length β2→1-fructans on prebiotic and immunomodulatory parameters in rats was studied [55]. In this study, β2→1-fructans supplementation increased the cecal lactobacilli and bifidobacteria counts and that IgA concentrations were increased in the order DP4 > DP8 > DP16. In addition, DP4, DP8, and DP16, but not DP23, increased IgA-producing plasma cells in the cecal mucosa. IFN-γ and IL-10 production in cecal CD4(+) T cells was enhanced solely by DP4. These results confirm that in vivo, chain length of β2→1-fructans is also of importance to the immunological response and the fact that these parameters were not correlated to any of the observed bacterial changes suggests that this is a direct effect on (immune) cells. This size exclusion effect for direct signaling of β2→1-fructans in vivo was previously suggested by Seifert and Watzl [8]. As there are many families of PRRs with typical carbohydrate binding properties and immunomodulatory capacities [56], we applied a strategy to target TLRs and NODs: as shown in this study, the activating capacity of β2→1-fructans on THP-1 cells was MyD88 dependent, and activation of TLR2 expressing HEK cells was strongly induced, which implies that signaling through TLRs, and specifically TLR2, is important in the immunomodulatory capacity of β2→1-fructans [11,27,28,57]. Other PRRs, i.e. C-type Lectin-like receptors or RIG-like receptors do not signal via MyD88, but utilize other mechanisms and molecules to transduce their signal [58,59].

CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT.

When studying β2→1-fructan mediated activation of HEK cells expressing individual TLRs or NODS, we observed that NF-κB/AP-1 was strongly induced via activation of TLR2, and to a lesser extent by TLR4, 5, 7, 8, and NOD2. No activation was observed for TLR3, 9, or NOD1. TLR3 does not signal to NF-κB/AP-1 via MyD88 but through its adapter Toll/interleukin-1 receptor homology-domain-containing adapter-inducing interferon-β (TRIF) [60]. However, activation by β2→1-fructans was virtually absent in the THP-1 DefMyD cells, indicating the importance of MyD88-related TLRs and no involvement of TLR3. TLR2 has many natural ligands of bacterial, fungal, viral, and even endogenous nature [61]. One of these is zymosan, a yeast PAMP consisting of β-glucans [62], which bear similarity to β2→1-fructans because both are prebiotic polysaccharides with β-glycosidic bonds. Although β-glucans are different molecules, it is tempting to speculate that the mechanism of TLR ligation by β2→1-fructans is similar to that of β-glucans, but this requires further biochemical studies. Another way of inducing different signals might be mechanistic differences in receptor interactions at the cellular surface. It is possible that the shorter chains only activate a few receptors at a time, and the receptors may be located relatively distant from each other, whereas the longer chain β2→1-fructans might show a property of clustering the relevant receptors on the membrane, thereby creating a molecular complex which enhances signal transduction or alters the downstream outcome. This clustering mechanism has been described for LPS, clustering substantial numbers of TLR4 [63] and may be a relevant mechanism for other TLRs as well. To our best knowledge this is the first study addressing the direct mechanism behind the immunomodulating capacity of specific β2→1- fructans, with the aim to get more insight into the protective associations attributed to dietary fibers in epidemiological studies [4]. We demonstrated the principle that β2→1-fructans possess direct signaling capacity on human immune cells, mainly through TLR2. These results suggest that direct TLR2 signaling events on immune cells could be part of the mechanism by which IL-10 production is induced in in vivo β2→1- fructan supplementation studies. Also we show structure-function relationships in vitro for β2→1-fructans which illustrate that caution should be taken in ascribing beneficial health effects to families of molecules with the similar structural features, as seemingly minor differences in chain length could induce opposite effects.

CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT.

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CHAPTER 2. IMMUNE MODULATION BY DIFFERENT TYPES OF β2→1-FRUCTANS IS TOLL-LIKE RECEPTOR DEPENDENT.

56. Delgado MA, Deretic V. (2009) Toll-like receptors in control of immunological autophagy. Cell Death Differ 16: 976-983. 57. de Kivit S, Kraneveld AD, Garssen J, Willemsen LE. (2011) Glycan recognition at the interface of the intestinal immune system: Target for immune modulation via dietary components. Eur J Pharmacol . 58. Kerrigan AM, Brown GD. (2010) Syk-coupled C-type lectin receptors that mediate cellular activation via single tyrosine based activation motifs. Immunol Rev 234: 335- 352. 59. Bruns AM, Horvath CM. (2012) Activation of RIG-I-like receptor signal transduction. Crit Rev Biochem Mol Biol 47: 194-206. 60. Kawai T, Akira S. (2010) The role of pattern-recognition receptors in innate immunity: Update on toll-like receptors. Nat Immunol 11: 373-384. 61. Oliveira-Nascimento L, Massari P, Wetzler LM. (2012) The role of TLR2 in infection and immunity. Front Immunol 3: 79. 62. Sato M, Sano H, Iwaki D, Kudo K, Konishi M, et al. (2003) Direct binding of toll-like receptor 2 to zymosan, and zymosan-induced NF-kappa B activation and TNF-alpha secretion are down-regulated by lung collectin surfactant protein A. J Immunol 171: 417-425. 63. Visintin A, Latz E, Monks BG, Espevik T, Golenbock DT. (2003) Lysines 128 and 132 enable lipopolysaccharide binding to MD-2, leading to toll-like receptor-4 aggregation and signal transduction. J Biol Chem 278: 48313-48320.

CHAPTER 3

Toll-like receptor 2 activation by β2→1 fructans protects barrier function of t84 human intestinal epithelial cells in a chain length- dependent manner

Leonie M. Vogt1, Diederick Meyer2, Gerdie Pullens3, Marijke M. Faas1,4, Koen Venema5, Uttara Ramasamy6, Henk A. Schols6, Paul de Vos1

1 Department of Pathology and Medical Biology, Division Medical Biology, Groningen University, University Medical Center Groningen 2 Sensus B.V., Borchwerf 3, 4704 RG Roosendaal 3 Cosun Food Technology Centre, Oostelijke Havendijk 15, 4704 RA, Roosendaal 4 Department of Obstetrics and Gynaecology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, the Netherlands 5TNO Quality of Life, Department of Biosciences, PO Box 360, 3700 AJ Zeist 6 Laboratory of Food Chemistry, Wageningen University, PO Box 8129, 6700 EV Wageningen

Published in J. Nutr. Vol. 144, p.1002-1008, Jul. 2014.

CHAPTER 3.TOLL-LIKE RECEPTOR 2 ACTIVATION BY β2→1-FRUCTANS PROTECTS BARRIER FUNCTION OF T84 HUMAN INTESTINAL EPITHELIAL CELLS IN A CHAIN LENGTH-DEPENDENT MANNER.

Abstract

Introduction: Dietary fiber intake is associated with lower incidence and mortality from disease, but the underlying mechanisms of these protective effects are unclear. We hypothesized that β2→1-fructan dietary fibers confer protection on intestinal epithelial cell barrier function via Toll-like receptor 2 (TLR2), and we studied whether β2→1-fructan chain-length differences affect this process. Methods: T84 human intestinal epithelial cell monolayers were incubated with 4 β2→1-fructan formulations of different chain-length compositions and were stimulated with the proinflammatory phorbol 12- myristate 13-acetate (PMA). Transepithelial electrical resistance (TEER) was analyzed by electric cell substrate impedance sensing (ECIS) as a measure for tight junction–mediated barrier function. To confirm TLR2 involvement in barrier modulation by β2→1-fructans, ECIS experiments were repeated using TLR2 blocking antibody. Results: After preincubation of T84 cells with short-chain β2→1- fructans, the decrease in TEER as induced by PMA (62.3 6 5.2%, P < 0.001) was strongly attenuated (15.2 6 8.8%, P < 0.01). However, when PMA was applied first, no effect on recovery was observed during addition of the fructans. By blocking TLR2 on the T84 cells, the protective effect of short- chain β2→1-fructans was substantially inhibited. Stimulation of human embryonic kidney human TLR2 reporter cells with β2→1-fructans induced activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), confirming that β2→1-fructans are specific ligands for TLR2. Conclusions: To conclude, β2→1-fructans exert time-dependent and chain length–dependent protective effects on the T84 intestinal epithelial cell barrier mediated via TLR2. These results suggest that TLR2 located on intestinal epithelial cells could be a target of β2→1-fructan– mediated health effects.

CHAPTER 3.TOLL-LIKE RECEPTOR 2 ACTIVATION BY β2→1-FRUCTANS PROTECTS BARRIER FUNCTION OF T84 HUMAN INTESTINAL EPITHELIAL CELLS IN A CHAIN LENGTH-DEPENDENT MANNER.

Introduction

3.1 Background It is becoming more accepted that dietary fiber intake leads to a reduced incidence of disease (1-4) and related mortality (5-8). The mechanisms behind this effect of dietary fibers are still incompletely understood. Factors that were suggested to play an important role are improvement of the gut microbiota composition and the related SCFA profiles in the intestine (9-11). However, direct immune effects by ligand interaction of the fibers with so-called pattern recognition receptors on gut immune cells (12-15) were also suggested. Another mechanism by which dietary fibers may contribute to health is by modulating the integrity of the intestinal epithelial barrier. A disrupted barrier is considered to play a role in the etiology and pathogenesis of several diseases (16), such as atopic eczema (17, 18), asthma (19, 20), inflammatory bowel disease (21, 22), diabetes (23-25), obesity (26, 27), celiac disease (28-30), and diarrhea-predominant irritable bowel syndrome (31, 32). A compromised intestinal barrier can be associated with hyperpermeability, also described as leaky gut syndrome (33). Disruption leads to increased translocation of bacteria, endotoxins, and other macromolecules, which can be important triggers for aberrant local or peripheral immune reactions (27, 34-36). A category of widely used dietary fibers is formed by β2→1-fructans. Depending on the polymer chain lengths, these fibers are also described as inulin, inulin-type fructans (ITFs), fructooligosaccharides, or oligofructose (10). β2→1- fructans are well studied for their beneficial effects on the gut microbiota and health (10, 37-41). Studies into the effects of β2→1-fructans on immune cells suggest that they exert direct effects by receptor–ligand interactions and subsequent cytokine production (15, 42). However, direct effects of β2→1-fructans on the barrier integrity of human intestinal epithelial cells are, to the best of our knowledge, not available. Previous studies from our group suggest that chain-length differences of β2→1- fructans should be taken into consideration when studying their signaling effects: shorter-chain β2→1-fructans induce a more anti-inflammatory cytokine profile in isolated human peripheral blood mononuclear cells compared with long-chain β2→1-fructans (15). Differences in chain length of β2→1-fructans may also induce different effects on epithelial cells. We investigated whether β2→1-fructans exert protective effects on barrier function of human intestinal epithelial cells. This was done by analyzing the effect of β2→1-fructans on transepithelial electrical resistance (TEER)

CHAPTER 3.TOLL-LIKE RECEPTOR 2 ACTIVATION BY β2→1-FRUCTANS PROTECTS BARRIER FUNCTION OF T84 HUMAN INTESTINAL EPITHELIAL CELLS IN A CHAIN LENGTH-DEPENDENT MANNER. of T84 intestinal epithelial cell monolayers, damaged with the barrier- disruptive agent phorbol 12-myristate 13-acetate (PMA). The dynamics of this process were studied with regard to timing of fiber incubation and fructan chain-length effects. Because β2→1-fructans were identified recently as Toll-like receptor (TLR) ligands (15) and TLR2 is highly important in intestinal barrier regulation (43), the other aim of this study was to investigate whether receptor interactions with TLR2 on the epithelial surface are involved in β2→1-fructan-mediated barrier modulation.

Methods

3.2 Experimental design T84 intestinal epithelial cells were grown to differentiated monolayer stage, and resistance across the monolayer was continuously measured at multiple frequencies after different challenges. Measurements performed at 500 Hz specifically represent the tight junction (TJ) mediated resistance and these were used to calculate the AUC. To establish whether β2→1- fructans exert protective or recovery effects, a damage model was introduced based on challenge of the T84 cells with a known barrier disrupting agent, PMA (44). The rationale for the first experiment was to study whether β2→1-fructans protect T84 cells against PMA-induced loss of TEER, and whether β2→1-fructan chain length profile is important in TEER modulation. T84 cells were incubated for 24h, with four different formulations of β2→1-fructans of different average DP and DP profile (ITF I–IV) provided by Sensus B.V., Roosendaal, the Netherlands, followed by addition of PMA (10 nM, Sigma-Aldrich Chemie B.V., the Netherlands). The second experiment was designed to investigate the possible time dependency of the effect of β2→1-fructan incubation. Cells were stimulated in different order with different compounds; protocol I) incubation with PMA for 6h followed by removal of the medium and addition of β2→1-fructans in culture medium at a final concentration of 100 μg/mL, or protocol II) incubation for 24h with β2→1-fructans, followed by addition of PMA. This stimulation medium was left on the cells for at least 6h to allow the PMA to take effect. As the interval for recovery in protocol I, a fixed period of time was taken (12h) following removal of PMA medium. This period was based on the average recovery time of the cells treated with PMA followed by control medium, represented by TEER values returning to the initial value (100%). As a

CHAPTER 3.TOLL-LIKE RECEPTOR 2 ACTIVATION BY β2→1-FRUCTANS PROTECTS BARRIER FUNCTION OF T84 HUMAN INTESTINAL EPITHELIAL CELLS IN A CHAIN LENGTH-DEPENDENT MANNER. measure for induced recovery, AUC was plotted for this time frame relative to the AUC of untreated control (without PMA), which was set to 100%. For protocol II, the interval for protection against the reduction of TEER was based on the time frame starting at the addition of PMA until the maximal decrease in TEER was reached (after 6h), and this fixed time frame was taken to calculate the AUC relative to untreated controls. With the third experiment, we aimed to study the role of TLR2 in β2→1-fructan mediated effects on T84 cells. To this end, T84 cells were incubated with culture medium or TLR2 blocking antibody (catalog # pab-hstlr2, 5 μg/mL, InvivoGen, Toulouse, France) for 10 min prior to addition of β2→1- fructans. Cells remained in this medium for 24h, followed by addition of PMA. The 6h time frame following PMA addition was used for AUC calculations. Finally, to confirm that β2→1-fructans signal through TLR2, HEK hTLR2 reporter cells were stimulated with ITFI-IV.

3.3 Investigational compounds To study the effects of β2→1-fructans on intestinal epithelial cell barrier function, four different formulations were applied, based on the chemical characterization of their chain length profiles by High-Performance Anion- Exchange Chromatography and High Pressure Size Exclusion Chromatography (15). Endotoxin concentrations in the formulations were analyzed by Toxikon (Leuven, Belgium), and all fell below 0.3 x 10-3 endotoxin units (EU) μg-1. Briefly, the average chain length of these formulations is as ITF I (Frutalose® OFP) < ITF II (Frutafit® CLR) < ITF III (Frutafit® HD) < ITF IV (Frutafit® TEX). ITF I is a fructooligosaccharide (FOS) compound, with chain lengths of ≤ DP10. ITF II is an inulin, enriched with FOS, with most chains shorter than DP10, but also containing chains with DPs up to 60. ITF III is ‘native’ inulin, and ITF IV can be described as a long chain enriched inulin. Both ITF III and IV consist predominantly of chains ranging from DP10 to DP60. Chain length profiles of the applied formulations are summarized in Figure 1.

3.4 T84 cell culture T84 human colon carcinoma cells (Sigma-Aldrich Chemie B.V., Zwijndrecht, the Netherlands) were grown to ca. 80% confluency at 37°C, 5% CO2 in culture medium consisting of 1:1 Ham’s F12 medium:DMEM, acquired premade from Sigma-Aldrich Chemie B.V.), supplemented with 10% HyClone FBS, Thermo Scientific, Breda, the Netherlands) and gentamicin (50 µg/mL, Life Technologies Europe B.V., Bleiswijk, the Netherlands). Cells were maintained as previously described (45). Trypsin was acquired from

CHAPTER 3.TOLL-LIKE RECEPTOR 2 ACTIVATION BY β2→1-FRUCTANS PROTECTS BARRIER FUNCTION OF T84 HUMAN INTESTINAL EPITHELIAL CELLS IN A CHAIN LENGTH-DEPENDENT MANNER.

Figure 1. Chain-length distribution of the applied fructan formulations. ITF I consists predominantly of fructan chains of DP3–DP10 and a small portion of monomers. ITF II consists mainly of fructan chains of DP3–DP10 but also contains chain lengths up to DP60 and a small portion of monomers. ITF III consists mainly of fructan chains of DP10–DP60, and a smaller portion is made up of fructan chains of DP3–DP10 and monomers. ITF IV comprises mostly chains longer than DP10, a small portion of DP3–DP10, and no monomers or dimers. DP, degree of polymerization; ITF, inulin-type fructan; W,weight.

MP Biomedicals, Eindhoven, the Netherlands, and EDTA (Titriplex III) from Merck Millipore, Amsterdam, the Netherlands.

3.5 Trans Epithelial Electrical Resistance measurements Multiple electrode gold-plated 8 well chamber slides (8W10E, Applied Biophysics, IBIDI, München, Germany) were coated with 400 µL/well of a 0.2% L-cysteine (Sigma-Aldrich Chemie B.V.) solution in PBS for 30 min at room temperature. Wells were washed twice with PBS, and coated overnight at room temperature with 400 µL/well of 1% PureColTM bovine tail collagen (Nutacon B.V., Leimuiden, the Netherlands) and 0.1% BSA (Sigma-Aldrich Chemie B.V.) in PBS. Wells were then washed twice with culture medium and cells were seeded at a density of 2x104 cells per well in a final culture volume of 400 μL/well. Prior to stimulation, the cells were maintained in the wells for 14 days to reach a stable TEER. Medium was changed twice a week. The chamber slides were put into an ECIS incubator (Z-Theta model, Applied Biophysics, Troy, New York, USA) and resistance was measured continuously at multiple frequencies (46).

CHAPTER 3.TOLL-LIKE RECEPTOR 2 ACTIVATION BY β2→1-FRUCTANS PROTECTS BARRIER FUNCTION OF T84 HUMAN INTESTINAL EPITHELIAL CELLS IN A CHAIN LENGTH-DEPENDENT MANNER.

3.6 HEK hTLR2 reporter cell culture and reporter assay HEK hTLR2 reporter cells were cultured and NF-κB activity was determined as previously described (15). As positive control, the additional TLR2 agonist heat killed Listeria monocytogenes was applied by adding 20 µl solution to 180 µl of cell suspension (1x108 bacteria/mL, InvivoGen).

3.7 Statistical analysis Statistical analysis was performed using GraphPad Prism 5.0 software. D’Agostino & Pearson omnibus normality test was used to test for normal data distribution. Statistical significance levels were determined by one- way ANOVA and Tukey’s Multiple Comparison test or by Dunnett’s Multiple comparison test to compare treatment with controls. Results are expressed as mean  SD. P-values < 0.05 were considered statistically significant.

Results

3.8 β2→1-fructans exert chain length dependent protection against PMA-induced loss of barrier function in T84 intestinal epithelial cells To study whether β2→1-fructans protect T84 cells against PMA-induced loss of TEER, and whether β2→1-fructan chain length profile is important in TEER modulation, T84 cells were incubated with four different formulations of β2→1-fructans of different average DP and DP profile, (ITF I–IV). AUC for the 6h time period following PMA addition was plotted for the different ITF treatments, as a percentage of the AUC of untreated control, which was set to 100 % (Figure 2). The damage model of PMA treatment induced a decrease in TEER, resulting in an AUC of 61.5 ± 5.8% (P<0.001) as compared to control. Strikingly, 24h of preincubation of T84 cells with 100 μg/mL of the ITF I or ITF II β2→1-fructans conferred a protective effect against PMA-induced loss of resistance (P<0.001 and P<0.01 respectively). ITF I conferred the strongest protection, with an AUC of 91.0 ± 6.6% of the control AUC, followed by ITF II, which rendered an AUC of 75.4 ± 3.2% of the control AUC. TEER values for treatment with the longer chain compounds (ITF III and IV) were not statistically different from TEER values as induced by PMA treatment, demonstrating that these compounds did not exert a protective effect. These results indicate that the protective effect of β2→1-fructans is a chain length dependent phenomenon, which is only conferred by the short chain formulations ITF I and ITF II.

CHAPTER 3.TOLL-LIKE RECEPTOR 2 ACTIVATION BY β2→1-FRUCTANS PROTECTS BARRIER FUNCTION OF T84 HUMAN INTESTINAL EPITHELIAL CELLS IN A CHAIN LENGTH-DEPENDENT MANNER.

Figure 2. Chain length–dependent protection of transepithelial electrical resistance across T84 epithelial cell monolayers treated with different ITF formulations and PMA. AUC was plotted for the time range starting at the addition of PMA and after a 6-h time period. Values are means ± SDs, n = 3. Data are representative of 3 individual experiments. Statistical significance levels were determined with 1-factor ANOVA and Tukey’s multiple comparison test. Labeled means without a common letter differ, P < 0.05. ITF, inulin-type fructan; PMA, phorbol 12-myristate 13-acetate.

3.9 The protective effect of short chain β2→1-fructans against PMA- induced T84 barrier loss is time dependent To establish if timing of short chain β2→1-fructan incubation is important in their functionality, two protocols for incubation were applied; I) preincubation of T84 cells with PMA for 6h followed by removal of the medium and addition of short chain β2→1-fructans (ITF I) in culture medium, and II) preincubation of T84 cells for 24h. with ITF I followed by addition of PMA (Figure 3). Figure 3A shows an example representative of relative TEER values obtained with protocol I, and the 12h interval used to calculate the AUC, which was subsequently plotted in figure 3B. Figures 3A and B show that protocol I did not induce TEER recovery effects as compared to PMA treatment. A representative example of relative TEER values obtained with protocol II, and the 6h interval used to calculate the AUC are plotted in figure 3C. Here, a significant protection was established, minimizing the decrease in TEER at 6h after PMA addition to 15.2 ± 8.8% (P<0.01), whereas PMA treatment alone induced a reduction of 62.3 ± 5.2% (P<0.001) of the initial TEER values. This protective effect was also observed when calculating the AUC (figure 3D). Over the 6h

CHAPTER 3.TOLL-LIKE RECEPTOR 2 ACTIVATION BY β2→1-FRUCTANS PROTECTS BARRIER FUNCTION OF T84 HUMAN INTESTINAL EPITHELIAL CELLS IN A CHAIN LENGTH-DEPENDENT MANNER.

Figure 3. Time-dependent impact of different incubation protocols on TEER across T84 epithelial cell monolayers. Example of TEER as induced by protocol I (A). AUC for 12 h from the time point of PMA addition, representing the period of recovery for PMA-treated cells to 100% of the initial value for protocol I (B). Values are means ± SDs, n = 6. Example of TEER as induced by protocol II (C). AUC for 6 subsequent hours after addition of PMA plotted as percentage of the AUC of untreated controls (D). Values are means ± SDs, n = 6. Data are representative of 3 individual experiments, and data for ITF I are shown for n = 6. Statistical significance levels were determined with 1-factor ANOVA and Tukey’s multiple comparison test. Labeled means without a common letter differ, P <0.05. ITF, inulin-type fructan; PMA, phorbol 12-myristate 13-acetate; TEER, transepithelial electrical resistance.

period, the AUC of PMA treatment was 69.5 ± 2.3% (P<0.001) of the AUC of untreated controls, whereas the AUC of cells pretreated with ITF I followed by PMA was 91.0 ± 6.6% (P<0.01) of the AUC of untreated controls. These results indicate that timing of incubation with β2→1- fructans is an important factor for protection of the T84 barrier function.

3.10 Blocking of TLR2 inhibits short chain β2→1-fructan mediated protection of TEER Because β2→1-fructans have recently been identified as TLR ligands (15), and TLR2 is highly important in intestinal barrier regulation (43), we hypothesized that the protective effect of short chain β2→1-fructans on epithelial cells against PMA might be mediated through TLR2. T84 cells

CHAPTER 3.TOLL-LIKE RECEPTOR 2 ACTIVATION BY β2→1-FRUCTANS PROTECTS BARRIER FUNCTION OF T84 HUMAN INTESTINAL EPITHELIAL CELLS IN A CHAIN LENGTH-DEPENDENT MANNER. were preincubated with normal culture medium or with TLR2 blocking antibody prior to 24h incubation with short chain β2→1-fructans, followed by PMA challenge (Figure 4). As the effects of longer chain β2→1-fructans did not induce protective effects, ITF III and IV were not included in the TLR2 blocking experiments. Figure 4A and B show representative examples of TEER values as induced by PMA, preincubation with IFT I and ITF II respectively, and pretreatment with the blocking antibody. AUC for a 6h time frame following PMA addition was plotted in figure 4C, showing that pretreatment with TLR2 blocking antibody significantly reduced the short chain β2→1-fructan mediated protection for both short chain formulations, with a TEER curve approaching the PMA curve. These results indicate that in T84 cells, TLR2 is involved in the protective mechanism of short chain β2→1-fructans against PMA.

Figure 4. Effects of blocking TLR2 on inulin-type fructan–mediated protection of T84 TEER. TEER of T84 cells with or without TLR2 blocking antibody (AB), incubation with ITF I for 24 h, and 10 nmol/L PMA (A). TEER of T84 cells with or without TLR2 blocking antibody (AB), incubated with ITF II for 24 h, and 10 nmol/L PMA (B). The AUC for 6 subsequent hours of each treatment (C). Data are representative of 3 individual experiments, and data for ITF I are shown for n = 9. Labeled means without a common letter differ, P < 0.05. Statistical significance levels were determined with 1-factor ANOVA and Tukey’s multiple comparison test. AB, antibody; ITF, inulin-type fructan; PMA, phorbol 12-myristate 13-acetate; TEER, transepithelial electrical resistance; TLR2, Toll-like receptor 2.

CHAPTER 3.TOLL-LIKE RECEPTOR 2 ACTIVATION BY β2→1-FRUCTANS PROTECTS BARRIER FUNCTION OF T84 HUMAN INTESTINAL EPITHELIAL CELLS IN A CHAIN LENGTH-DEPENDENT MANNER.

Figure 5. NF-kB/AP-1 activity of HEK human TLR2 reporter cells. Fold induction for positive controls represented by 108 cells/mL heatkilled Listeria monocytogenes, and TLR agonist FSL-1, and a concentration range of inulin-type fructans (milligrams per liter) was plotted as compared to unstimulated control (medium, set to 1). Values are means ± SDs, n = 3. Statistical significance levels were determined with Dunnett’s multiple comparison test. *Labeled means are different from control, P < 0.05. AP-1, activator protein 1; FSL-1, Pam2CGDPKHPKSF, synthetic lipoprotein derived from Mycoplasma salivarium; HEK, human embryonic kidney; HKLM, heat-killed Listeria monocytogenes; ITF, inulin-type fructan; TLR2, Toll-like receptor 2.

3.11 β2→1-fructans exert TLR2-mediated NF-κB activation in HEK hTLR2 reporter cells TLR2 was suggested as a mediator of β2→1-fructan signaling in immune cells (15), and in previous studies an important role has been attributed to TLR2 in modulating barrier function (43, 47-49). To confirm the role of TLR2 in β2→1-fructan signaling, HEK hTLR2 reporter cells were incubated with a concentration series of β2→1-fructans of different chain lengths. β2→1-fructans induced TLR2-mediated NF-κB activation in the reporter cell line (Figure 5). Short chain β2→1-fructans (ITF I) induced a 2.8 ± 1.2 fold induction (P<0.05) of NF-κB/AP-1 activation compared with control, and with increasing mean fructan chain length, the fructans conferred stronger activation, up to 5.9 ± 0.5 fold induction (P<0.05) for the longest mean fructan chain formulation (ITF IV) as compared to control. These results confirm the role of TLR2 in β2→1-fructan signaling and indicate that fructan chain length is an important factor in determining the strength of the TLR2 response. Short chain β2→1-fructans conferred a moderate TLR2 activation, whereas the longer chain β2→1-fructans induced a relatively strong TLR2 response.

CHAPTER 3.TOLL-LIKE RECEPTOR 2 ACTIVATION BY β2→1-FRUCTANS PROTECTS BARRIER FUNCTION OF T84 HUMAN INTESTINAL EPITHELIAL CELLS IN A CHAIN LENGTH-DEPENDENT MANNER.

Discussion

To our best knowledge, this is the first time β2→1-fructans have been identified in their capacity as modulators of human intestinal epithelial cell barrier function through TLR2. Proof of principle was established that β2→1-fructan dietary fibers can protect the integrity of intestinal epithelial cell monolayers. This effect was observed for the shorter chain ITFs (ITF I and II) but not for the longer chain formulations (ITF III and IV). This is in accordance with a previous study in our group, in which chain length differences of the applied β2→1-fructans were important factors in inducing different effects on human immune cells with regard to cytokine profiles (15). In this study, the short chain β2→1-fructans induced a more anti-inflammatory cytokine pattern compared to the longer chain formulations, indicating chain length dependent differences in downstream effects in immune cells. In addition, two studies by Ito et al. performed in rats corroborated chain length dependent effects on immune cells as well as intestinal barrier function in vivo. The studies by Ito et al. demonstrated that β2→1-fructans stimulated intestinal immune parameters in a chain length dependent manner (50), and that short chain β2→1-fructans reduced translocation of endotoxins and bacteria in a TNBS-induced colitis model (51). By using different timing protocols regarding short chain β2→1- fructan or PMA treatment we observed a protective effect of the fructans on TEER, provided that the cells were incubated with the fructans for 24h, before stimulation with PMA. Treatment with PMA followed by incubation with β2→1-fructans did not induce recovery effects, indicating that in this model, fructans exerted protective effects rather than effects on repair processes. This conclusion was based on the described model using low dose treatment. The rationale behind treatment with low dose fructans (100 µg/ml) was based on our previous results in peripheral blood mononuclear cells (15), where this dose already induced substantial cell activation in the form of production of several cytokines upon 24h of stimulation. In addition, several studies have shown that preincubating cells of different tissue types with TLR2 agonists can protect against detrimental effects of barrier disruptive agents or ischemia-reperfusion injury (47-49, 52, 53). In these studies, protection is often established under low dose stimulatory conditions. With the current study, novel proof of principle was demonstrated for time dependent protective effects of β2→1-fructans on barrier function of human intestinal epithelial cells. Whether the critical time frame of

CHAPTER 3.TOLL-LIKE RECEPTOR 2 ACTIVATION BY β2→1-FRUCTANS PROTECTS BARRIER FUNCTION OF T84 HUMAN INTESTINAL EPITHELIAL CELLS IN A CHAIN LENGTH-DEPENDENT MANNER. preincubation with β2→1-fructans can be reduced as compared to the 24h preincubation protocol while retaining a protective effect remains to be studied. This time frame may give an indication which sort of cellular processes are affected, such as receptor/adapter molecule assembly at the cell membrane, modulation of kinase activity, or further downstream effects such as nuclear translocation of messenger molecules, gene transcription, and protein expression. These results prompt further studies into the exact mechanisms behind the observed protective effects of the fructans on the epithelium. Considering epithelial cells of the intestine specifically, TLR2 is an important player in regulating permeability and thus barrier function (43). The role of TLR2 in the protective action of β2→1-fructans on intestinal epithelial cells was confirmed with blocking experiments. In addition, the specific dynamics of β2→1-fructan mediated TLR2 activation were demonstrated. Besides possible fructan chain length-, or dose dependent effects on the strength of TLR2 activation in intestinal epithelial cells, differences in TEER modulation may be due to the ability of TLR2 to heterodimerize with TLR1 (54), TLR6 (54) or TLR10 (55), depending on the stimulus (43, 54), and the ability to signal together with a spectrum of different coreceptors (56). These features of TLR2 diversify the downstream effects of TLR2, and may provide an explanation for chain length induced differences between the different fructan formulations. The discrepancy between the longer chains inducing strong TLR2 activation but not exerting a protective effect on barrier function, could be due to mechanistic differences in receptor interactions at the cellular surface (15). This type of mechanism has been previously described for TLR4 by Visintin et al. (57). They suggest that differences in agonist clustering mechanisms for TLRs culminate in enhanced signal transduction and different downstream reactions. This is in accordance with our previous observations that inulin-type fructans have different TLR activation patterns (15). We suggest that by activating different numbers of receptors at a time, and at different distance from each other on the cell membrane, long chain β2→1-fructans may induce a different cellular response than the short chain β2→1-fructans. However an in depth discussion of these pathways is beyond the scope of the present study, and the role of TLR2 dynamics in barrier function is subject of further research in our lab. As an analogue of the endogenous second messenger diacylglycerol, PMA induces activation and translocation of protein kinase C (PKC), leading to elevated intracellular Ca2+ and modulation of TEER (44). PMA

CHAPTER 3.TOLL-LIKE RECEPTOR 2 ACTIVATION BY β2→1-FRUCTANS PROTECTS BARRIER FUNCTION OF T84 HUMAN INTESTINAL EPITHELIAL CELLS IN A CHAIN LENGTH-DEPENDENT MANNER. challenge of T84 cells can be viewed as a simplified model for dietary stimuli (58) or intestinal pathogens (59) involved in modulating PKC signaling and affecting the gut barrier. The read out for TLR2 activation in the HEK reporter cell line was analyzed by the production of SEAP upon NF-κB activation. In intestinal epithelial cells, TLR2 activation was shown to acts as an NF-κB inhibitor (43). As PMA can also target NF-κB, downstream of PKC-β1, via stabilization of IκBα (60, 61), inhibition of NF- κB could be one of the downstream mechanisms by which the epithelial barrier is partially protected by the fructans. Although PKC was not studied in our experiments, results from our model suggest that short chain β2→1-fructans may interfere in this pathway, by partly inhibiting the cellular response to PMA. Previous studies with inulin have shown that their signaling can induce PKC activation in RAW 264.7 cells (60) and in rat distal colonic mucosa (62). However, the typical PKC dynamics are greatly dependent on the type of tissue and the organism which is studied (44). Moreover, the PKC family consists of different subclasses and several isoforms, each having distinct dynamics for activation and downstream effects (63-67). A study in T84 cells by Song et al. (68), showed that PMA induced PKCα translocation to the apical surface, which was correlated with the a decreased TEER. Interestingly, PKCα activation has previously been linked to TLR2 signaling cascades in mouse and human dendritic cells (69). In conclusion, β2→1-fructans may protect the integrity of the intestinal barrier from damage, by directly binding to TLR2. Since they also have immunomodulating effects and prebiotic effects, they form a promising category of dietary fibers with regard to intestinal health effects, and further studies into their effects upon ingestion on physiological, cellular, and molecular level are warranted.

CHAPTER 3.TOLL-LIKE RECEPTOR 2 ACTIVATION BY β2→1-FRUCTANS PROTECTS BARRIER FUNCTION OF T84 HUMAN INTESTINAL EPITHELIAL CELLS IN A CHAIN LENGTH-DEPENDENT MANNER.

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CHAPTER 4

Cellulose alters the expression of NF-κB-related genes and TLR-related genes in human peripheral blood mononuclear cells

Leonie M. Vogt1*, Mark V. Boekschoten2, Philip J. de Groot2, Marijke M. Faas1,3, Paul de Vos1

1Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands 2Division of Human Nutrition, Wageningen University, Wageningen, the Netherlands 3 Department of Obstetrics and Gynaecology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, the Netherlands

Journal of Functional Foods, in press.

CHAPTER 4. CELLULOSE ALTERS THE EXPRESSION OF NF-κB RELATED GENES AND TLR-RELATED GENES IN HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS.

Abstract

Scope: There is renewed interest to apply fibers from root vegetable byproduct for health promoting nutrition. In this study, the immunomodulatory and epithelial barrier effects of cellulose were evaluated. Methods and Results: Nuclear factor kappa B (NF-κB) activation was studied using reporter assays. Human peripheral blood mononuclear cells (PBMCs) were stimulated with cellulose followed by microchip gene expression analysis. Resistance of T84 intestinal epithelial cells was measured upon phorbol 12-myristate 13-acetate (PMA)-induced damage in the presence and absence of cellulose. Reporter assays confirmed activation through TLR/MyD88 dependent-, and independent pathways. Cellulose induced upregulation of three NF-κB related genes, i.e. cluster of differentiation 40 (CD40) molecule, interleukin 1 receptor antagonist (IL- 1Ra), and interleukin-1 receptor-associated kinase 1 (IRAK1). Five upregulated genes related specifically to TLR signaling were identified, i.e. interleukin 1 receptor antagonist (IL-1Ra), interleukin-1 receptor- associated kinase 1 (IRAK1), jun proto-oncogene, mitogen-activated protein kinase kinase 3 (MAP2K3), and mitogen-activated protein kinase 13 (MAPK13). Cellulose did not improve or protect T84 resistance. Conclusion: Cellulose does not directly affect intestinal cell barrier function. However, it alters gene expression in human immune cells and activates TLR and non-TLR related pattern recognition pathways, indicating the immunomodulatory potential of cellulose as major component of root pulp byproduct.

CHAPTER 4. CELLULOSE ALTERS THE EXPRESSION OF NF-κB RELATED GENES AND TLR-RELATED GENES IN HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS.

Introduction

4.1 Chicory root pulp and cellulose In the current society, sustainability and health are considered important assets. The health benefits of dietary fibers are becoming more evident, leading to a renewed interest in root pulp byproducts for application in the functional food industry. Root vegetables, such as chicory and sugar beet, are valuable sources of mono-, oligo-, and polysaccharides, which are used in the food and feed industry [1,2]. The oligo-, and polysaccharides are prebiotic fibers, as they can stimulate numbers and activity of intestinal bifidobacteria and lactobacilli upon consumption [3,4]. After extraction of sucrose and fructose, the remaining fraction of these root vegetables can be used as a high fiber livestock feed [5,6]. One of the major compounds of chicory root [7] and beet root pulp [8] is cellulose. This cell wall polysaccharide consists of a linear chain with a varying number of β(1→4) linked D-glucose units with the formula

(C6H10O5)n [9,10]. Cellulose is water insoluble and inert to digestive enzymes in the human small intestine [11,12], but it is better known for its fecal bulking properties and moderate fermentability in the large intestine, leading to production of short chain fatty acids [13]. Due to these characteristics cellulose can be referred to as a dietary fiber [13]. As several types of dietary fibers induce immune signaling by directly binding to innate immune receptors [14-16], our aim was to study whether cellulose induces similar effects. By incubating Toll-like receptor (TLR) reporter cells with cellulose, nuclear factor kappa B (NF-κB)-, and TLR activation as well as TLR/Myeloid Differentiation Factor 88 (MyD88) independent activation was investigated. Peripheral blood mononuclear cells are often applied as a model to study the immunogenic potential of dietary substances such as probiotics or prebiotics [16-18]. By analyzing cellulose-induced gene expression of isolated peripheral blood mononuclear cell (PBMCs) we aimed to identify genes which are differentially regulated upon direct contact with cellulose. In addition, evidence is emerging that dietary fibers can be beneficial for intestinal epithelial barrier function by activation of certain innate immune receptors of intestinal epithelial cells [19-23]. By analysis of the resistance across T84 human intestinal epithelial cell monolayers we studied whether addition of cellulose could improve the barrier function. Furthermore, a damage model was applied to study possible protective effects. By preincubation of the cells with cellulose, we studied whether a phorbol ester-induced decrease in trans epithelial electrical resistance (TEER) could

CHAPTER 4. CELLULOSE ALTERS THE EXPRESSION OF NF-κB RELATED GENES AND TLR-RELATED GENES IN HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS. be prevented or partially inhibited. These experiments were aimed to evaluate whether besides its bulking properties and the stimulation of SCFA in the intestine, cellulose has the capacity to directly induce activation of immune cells and stimulate or protect the intestinal barrier function.

Methods

4.2 Investigational compound As an investigational compound to study the effects of well-characterized and pure cellulose, we used commercially available cellulose (Sigmacell® highly purified fibers, Type 101, Sigma-Aldrich Chemie B.V., Zwijndrecht, the Netherlands). Cellulose consists of a linear chain with β(1→4) linked D- glucose subunits, characterized by the chemical formula (C6H10O5)n [9,10]. An example of a Haworth projection of this molecule is depicted in Figure 1. Endotoxin content (endotoxin units, EU) of cellulose was assessed by Toxikon (Leuven, Belgium) and was 119 EU/g. This concentration has minimal effects on the responsiveness of the applied cell types.

4.3 Ethical statements Isolation of PBMCs from human volunteers was conducted within the University Medical Center Groningen, in the Netherlands. Written informed consent from the volunteers was obtained, and data were analyzed and presented anonymously. This research and consent procedure have been approved by the ethical review board of the University Medical Center, Medisch Ethische Toetsingscommissie University Medical Center Groningen, as documented in the approved application ‘‘2007/255’’. All clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki.

4.4 Cell culture and assays reporter cell lines The following reporter cell lines were acquired from InvivoGen, (Toulouse, France) : THP1-XBlue with inserted constructs for MD2, CD14, and secreted embryonic alkaline phosphatase (SEAP); THP1-Xblue-defMyD reporter cells with inserted constructs for SEAP, and deficient in MyD88 activity; Human embryonic kidney (HEK-Blue) cells with inserted constructs for SEAP and human TLR2, and Human embryonic kidney (HEK- Blue) cells with inserted constructs for SEAP and human TLR4. These cell lines were cultured and maintained as previously described [16]. NF-κB

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Figure 1. Haworth projection of cellulose [87]

activity of THP1 cell lines and HEK hTLR reporter cells by 24 h of stimulation with cellulose (dose range 100 µg/mL - 2 mg/mL) was determined as previously described [16].

4.5 Human Peripheral Blood Mononuclear Cell isolation, culture, and stimulation PBMCs of ten healthy male volunteers were isolated and cultured as previously described [16]. PBMCs were counted using a Z2 cell and particle counter (Beckman Coulter Nederland B.V., Woerden, the Netherlands) and 2 x 106 cells per well were transferred to a 24 wells plate in a total volume of 1 ml per well. As a model for direct interaction of cellulose with immune cells [16-18], PBMCs were incubated for 24 h (37°C and 5% CO2) with a thoroughly homogenized suspension of 1 or 100 µg/mL of cellulose (Sigma-Aldrich Chemie B.V.), or with normal culture medium, or with 2 ng/mL LPS ((Escherichia coli serotype 0111:B4; Sigma-Aldrich Chemie B.V.).

4.6 PBMC RNA isolation After stimulation, PBMCs were harvested and transferred to sterilized 1.5 mL eppendorf containers, centrifuged for 5 min at 300 g, resuspended in RNAprotect Cell Reagent (Qiagen, Venlo, the Netherlands) and stored at - 20°C. RNA was isolated using RNeasy Plus Mini Kit (Qiagen) according to the following protocol. PBMC suspension in RNAprotect was centrifuged

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CHAPTER 4. CELLULOSE ALTERS THE EXPRESSION OF NF-κB RELATED GENES AND TLR-RELATED GENES IN HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS. as described above, supernatant was discarded and cell pellet was homogenized in 350 µL of 0.143 M β-mercaptoethanol / RLT buffer Plus by passing the lysate through a 20-gauge needle with an RNase-free syringe 5 times. Homogenized lysate was transferred to gDNA Eliminator spin columns and centrifuged for 30 s at 10.000 g. 350 µL of 70% ethanol was added to the flow-through and samples were mixed by pipetting. 700 µL of the sample was transferred to an RNeasy spin column and centrifuged for 15 s at 10.000 g. Flow-through was discarded and columns were washed once with 700 µL of RW1 buffer and once with 500 µL of RPE buffer with centrifuge steps of 15 s at 10.000 g and discarding flow- through after each centrifugation step. After washing the columns for a second time with 500 µL of RPE buffer and centrifuging for 2 min at 10.000 g, spin columns were transferred to fresh collection tubes and 30 µL of RNase-free water was directly added to the column membrane. Columns were centrifuged for 1 min at 10.000 g to elute the RNA. RNA quality was checked using an Agilent 2100 bioanalyzer (Agilent Technologies, Amsterdam, the Netherlands).

4.7 Microchip analysis Microchip analysis of PBMC gene expression was performed at the University Medical Center Groningen, the Netherlands. cDNA and cRNA synthesis were performed with an Ambion Illumina TotalPrep-96 RNA Amplification kit (Life Technologies, Bleiswijk, the Netherlands) following the manufacturer's instructions. PBMC cRNA (300 ng) was labeled using an Epicentre TargetAmp Nano-g Biotin-aRNA Labeling kit (Westburg BV, Leusden, the Netherlands) and hybridized to Illumina Sentrix® HumanHT- 12 v3 whole genome microarray BeadChips coding over 25.000 genes, (Illumina, San Diego, USA). Sample labeling, chip hybridization, and image scanning were performed according to the manufacturer's instructions. All arrays met our criteria of the performed quality control. The Bioconductor Lumi package was used for the quality check and normalization of the Illumina arrays [24,25]. The lumiR command was used to load the text-file as generated by the Beadstudio software. The NuIDs were automatically generated upon loading the text-file [26]. The quality was checked by inspecting density plots, boxplots, array-array correlation plots, and the output of the lumiQ command [25]. The normalization procedure was done in 2 steps. First, the Variance Stabilization Transformation (VST) was applied followed by the Loess normalization procedure as it is available in the lumiN function [27]. The normalized data was used for doing the statistics utilizing the Limma

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CHAPTER 4. CELLULOSE ALTERS THE EXPRESSION OF NF-κB RELATED GENES AND TLR-RELATED GENES IN HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS. package [28]. Pathway analysis was performed with Ingenuity Pathways Analysis software (Qiagen, Venlo, The Netherlands).

4.8 T84 cell culture and epithelial cell barrier measurements T84 human colon carcinoma cells (Sigma-Aldrich Chemie B.V) were grown to ca. 80% confluency at 37°C, 5% CO2, in culture medium consisting of 1:1 Ham’s F12 medium : DMEM, acquired premade from Sigma-Aldrich Chemie B.V.), supplemented with 10% HyClone fetal bovine serum (FBS, Thermo Scientific, Breda, the Netherlands) and gentamicin (50 µg/mL, Life Technologies Europe B.V.). Cells were maintained as previously described [29]. Trypsin was acquired from MP Biomedicals, Eindhoven, the Netherlands, and EDTA (Titriplex III) from Merck Millipore, Amsterdam, the Netherlands. Multiple electrode gold-plated 96 well chamber slides (96W20idf, Applied Biophysics, IBIDI, München, Germany) were coated with 300 µL/well of a 0.2% L-cysteine (Sigma-Aldrich Chemie B.V.) solution in DMEM (Life Technologies Europe B.V.) for 30 min at room temperature. Wells were washed twice with DMEM, and coated overnight at room temperature with 300 µL/well of 1% PureColTM bovine tail collagen (Nutacon B.V., Leimuiden, the Netherlands) and 0.1% BSA (Sigma-Aldrich Chemie B.V.) in culture medium. Wells were then washed twice with culture medium and cells were seeded at a density of 2 x 105 cells per well in a final volume of 300 μL/well. Prior to stimulation, the cells were maintained in the wells for 14 days to reach a stable TEER, and medium was changed every other day. Resistance was measured continuously at multiple frequencies [30] upon placing the chamber slides in an electric cell substrate impedance sensing (ECIS) incubator (Z-Theta model, Applied Biophysics, Troy, New York, USA). Measurements performed at 1000 Hz were used to calculate the area under the curve (AUC), as data acquired at relatively low frequency values specifically represent the tight junction mediated resistance in the 96W20idf plates. To establish whether cellulose exerts protective effects, a damage model was applied based on T84 cell incubation with phorbol 12-myristate 13-acetate (PMA, 10nM, Sigma-Aldrich Chemie B.V.), which is a barrier disrupting agent [31]. Briefly, T84 cells were incubated with a concentration series of thoroughly homogenized cellulose in culture medium (1µg/mL – 2mg/mL) for 24 h, followed by addition of PMA. Cells were maintained in this stimulation medium for 24 h and this time frame was subsequently used to calculate the AUC relative to untreated controls.

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4.9 Statistical analysis Analysis of the microchip array data was performed using Genespring GX 9 software (Agilent Technologies, Santa Clara, USA). Genes were defined as significantly changed when the False Discovery rate q-value of the intensity-based moderated t-statistics was < 0.01 and fold change > 1.2. Statistical analysis of the reporter cell data and the ECIS data was performed by Wilcoxon Signed Rank test. Significant differences as compared to control (p < 0.05) are indicated with an asterisk.

Results

4.10 Cellulose activates NF-κB via TLR2 and TLR4 To gain insight in the TLR dependency of the effects of cellulose on PBMCs, activation of THP1 and HEK-Blue TLR reporter cells by cellulose was studied (Figure 2). THP1 cell lines express all TLRs [32]. By studying the activation of THP1 MD2-CD14 cells and comparing this with activation of THP1 DefMyD cells that express a non-functional MyD88, it is possible to determine the TLR dependency of the responses [16]. In THP1 MD2- CD14 cells, cellulose induced significant activation of NF-κB as compared to control, at the doses of 400 and 2000 µg/mL (Figure 2A). Subsequently, to confirm whether this NF-κB activation was mediated by TLRs, THP1 DefMyD cells were incubated with cellulose. In this cell line also significant NF-κB activation was observed as compared to control, suggesting that the activation of NF-κB in THP1 MD2-CD14 cells was partly mediated through TLR/MyD88-dependent and partly through TLR/MyD88- independent signaling pathways (Figure 2B). As TLR2 and TRL4 are important TLRs which recognize dietary carbohydrate oligomers and polymers [16,33-36], we investigated whether NF-κB activation by cellulose was mediated via TLR2 or TLR4. To this end, HEK-Blue TLR reporter cells with individual TLR constructs were stimulated with cellulose. As depicted in Figure 2C, cellulose induced statistically significant TLR2-mediated NF-κB activation at 400, 1000, and 2000 µg/mL. The dose-related pattern of TLR4-mediated NF-κB activation appeared to be biphasic (Figure 2D), with the low doses inhibiting NF-κB, and cellulose at a dose of 2000 µg/mL inducing a small increase in activation (1.2 fold as compared to control, p < 0.05).

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Figure 2. Reporter cell assays for TLR mediated NF-κB activation. A) THP1 MD2-CD14 cells incubated with control medium, endotoxin free water, cellulose, and LPS (100 ng/mL). B) THP1 DefMyD cells incubated with control medium, endotoxin free water, cellulose, and Tri- DAP (L-Ala-γ-D-Glu-mDAP, 25 μg/mL). C) HEK-Blue hTLR2 cells incubated with control medium, endotoxin free water, cellulose, and heat-killed Listeria monocytogenes (HKLM, 1*108 cells/mL). D) HEK-Blue hTLR4 cells incubated with culture medium, endotoxin free water, cellulose, and LPS (100 ng/mL, n=8).

4.11 Cellulose alters the expression of NF-κB and TLR-related genes in human peripheral blood mononuclear cells To gain insight in the pathways in addition to the TLR-dependent pathways that cellulose activates, we performed microarray analysis on cellulose exposed human PBMCs. PBMC RNA was isolated and hybridized to whole-genome expression microarrays. Quality control of the hybridizations and primary data analysis were performed as previously described [37] to ensure that the array data were of the highest possible quality. The in vitro stimulation of human PBMCs with cellulose resulted in the differential expression of a number of genes as compared to unstimulated controls. To specifically target genes related to NF-κB-, and TLR-signaling, comparisons and pathway reconstruction of these transcriptional networks were made using an in silico approach.

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Significant changes in expression of 8 genes related to NF-κB-signaling and 9 genes related to TLR-signaling induced by cellulose are visualized in Figures 3 and 4 respectively. Within the NF-κB pathway, the following 3 genes were upregulated: CD40 molecule (Tumor Necrosis Factor receptor superfamily member 5), interleukin 1 receptor antagonist, and interleukin- 1 receptor-associated kinase 1. In this pathway, 5 genes were downregulated, i.e. ataxia telangiectasia mutated (ATM serine/threonine kinase),phosphatidylinositol-4,5-bisphosphate 3-kinase, phosphoinositide- 3-kinase regulatory subunit 1 (alpha), TLR5, and TLR7. Within the TLR signaling cascade, 5 genes were upregulated by cellulose. These were interleukin 1 receptor antagonist, interleukin-1 receptor-associated kinase 1, jun proto-oncogene, mitogen-activated protein kinase kinase 3, and mitogen-activated protein kinase 13. The 4 genes which were downregulated by cellulose were CD14 molecule, mitogen-activated protein kinase kinase kinase 1, TLR5, and TLR7. Results are summarized in Tables 1 and 2. As a positive control for NF-κB-, and TLR-signaling, gene expression of LPS-stimulated PBMCs was analyzed. Significant changes in expression of 24 genes related to NF-κB-signaling and 19 genes related to TLR-signaling were induced (Figures 5 and 6). Results are summarized in Tables 3 and 4.

4.12 Results TEER TEER measurements showed that preincubating differentiated monolayers of T84 human intestinal epithelial cells with different doses of cellulose did not significantly protect the cells against a PMA-induced decrease in TEER (Figures 7A and B). The 2000 µg/mL dose appeared to induce a small ameliorating effect, however the AUC was not significantly different from the condition with PMA only. Addition of cellulose only to T84 cell monolayers also did not change the TEER as compared to addition of control medium (Figure 7).

Discussion

In this study, effects of cellulose on immune cells and intestinal epithelial cells were studied to obtain insight into the potential health promoting value of cellulose, as major component of root pulp byproduct. We hypothesized that cellulose can directly activate immune cells through

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TLRs as reported for other dietary fibers [14-16]. However, our results show both TLR/MyD88-dependent as well as TLR/MyD88-independent

Figure 3. Ingenuity Pathway Analysis showing significantly up-, and down regulated molecules within NF-kB signaling pathways upon stimulation of human PBMCs with cellulose, as compared to control. Upregulated genes are indicated in red, downregulated genes are indicated in green. activation. Not all TLRs utilize MyD88 as adapter, TLR3 signals via the adapter molecule TRIF [38]. TLR3 could thus be one of the PRRs which mediated the MyD88 independent NF-κB activation as measured in the THP1 DefMyD cells. However, this type of activation could also have occurred through other MyD88-independent PRRs expressed by the THP1 cells, such as c-type lectins [39]. Unfortunately, there are many c-type

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CHAPTER 4. CELLULOSE ALTERS THE EXPRESSION OF NF-κB RELATED GENES AND TLR-RELATED GENES IN HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS. lectins and no reporter cells yet to identify their specific ligands. We were able to show that TLR2 and TLR4 are involved. The activation as elicited in TLR2 reporter cells occurred at the doses of 400 and 2000 µg/mL, and only showed weak activation as compared to the natural pathogen associated ligand (heat killed Listeria monocytogenes) which was used as a positive control. Compared to activation levels reported for other fiber ligands such as inulin-type fructans this activation is also relatively weak [16]. However, we still feel that this TRL2 activation can occur in vivo, as in a normal Western diet, the average estimated daily intake is 3.2 g [40], which could provide concentrations of cellulose in the intestine that are within the range of the applied doses in these experiments. PBMCs are often used to study immune effects of bioactive food components [16,41,42] as they express many PRRs, including TLRs and c- type lectins. The focus of this study was on the analysis of NF-κB-related changes in gene expression upon stimulation with cellulose in order to gain insight in the pathways which are activated by cellulose.

Figure 4. Ingenuity Pathway Analysis showing significantly up-, and down regulated molecules within TLR signaling pathways upon stimulation of human PBMCs with cellulose, as compared to control. Upregulated genes are indicated in red, downregulated genes are indicated in green.

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Table 1. Fold induction of up-, and downregulated genes in the NF-κB pathway by cellulose.

Table 2. Fold induction of up-, and downregulated genes related to TLR signaling by cellulose.

Several genes were affected in their expression, demonstrating that cellulose induces transcriptional changes in several innate immune pathways in human PBMCs, as will be discussed below. The differentially regulated genes by cellulose encode molecules of several functional categories. In respective order of strength of induction the upregulated genes within the NF-κB pathway were IL-1Ra,

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IRAK1, and CD40. IL-1Ra is an interleukin receptor antagonist, which is expressed as a secreted isoform from monocytes, macrophages, neutrophils, and other cells (sIL-1Ra), or as an intracellular isoform (icIL- 1Ra1, 2, or 3) [43]. Il-1Ra competitively inhibits IL-1 binding to cell-surface receptors, and the balance between IL-1 and IL-1Ra is important in preventing the development or progression of several inflammatory diseases [43]. IRAK1 is one of two putative serine/threonine kinases that become associated with the interleukin-1 receptor (IL-1R) upon stimulation. It is rapidly recruited by MyD88 to the receptor-signaling complex upon TLR activation [44-46], leading to phosphorylation and kinase activation by IRAK4 [47,48]. IRAK1 plays a critical role in initiating innate immune responses against foreign pathogens [49]. CD40 protein is a costimulatory receptor molecule, which is expressed by antigen presenting cells such as dendritic cells, macrophages, and B cells, and also by T cells [50,51]. It is required for their proliferation [52], maturation [53- 55], and effective immune reactions [54], including induction of chemokines and T helper 1 skewing cytokines [52,56]. In addition, CD40/CD40L interaction of CD4+ T cells and CD8+ T cells is fundamental for induction of CD8+ T cell memory [51,57]. Upregulation of these genes as observed in PBMCs, could thus represent a stimulatory effect on immune status by induction of immune cell recruitment and activation, antigen presentation processes, and subsequent adaptive immune responses. However, not only upregulated but also downregulated genes within the NF-κB pathway were observed. Ataxia telangiectasia mutated (ATM) plays a role in cell cycle delay after DNA damage [58]. It has a broad range of substrates related to DNA repair, apoptosis, G1/S transition, intra- S checkpoint and G2/M checkpoints, gene regulation, translation initiation, and telomere maintenance [59]. PIK3cG is involved in inflammatory and allergic responses [60,61]. It is thought to play a role in the regulation of development, proliferation, migration, and function of B cells, T cells, and NK cells [62-64], and controls motility of dendritic cells. Its regulatory subunit, PIK3R1 which was also downregulated, is necessary for the insulin-stimulated increase in glucose uptake and glycogen synthesis in insulin-sensitive tissues [65]. This protein is tightly involved in regulation of PIK3cG in B cells and T cells [66]. Some TLRs were downregulated. TLR5 is an innate receptor which recognizes bacterial flagellin [67], and TLR7 is an innate receptor which recognizes single-stranded RNA in endosomes, represented by viral material which is internalized by macrophages and dendritic cells [68]. Another downregulated gene within the TLR pathway, not yet mentioned in the NF-κB pathway is CD14. CD14 protein acts as a

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CHAPTER 4. CELLULOSE ALTERS THE EXPRESSION OF NF-κB RELATED GENES AND TLR-RELATED GENES IN HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS. co-receptor, along with TLR4 and MD-2, in the detection of LPS and LTA (lipoteichoic acid) in the presence of lipopolysaccharide-binding protein (LBP) [69,70]. Downregulation of these genes within the NF-κB-, end TLR- pathways could be part of a negative feedback loop to protect against overstimulation and unwanted reactions of immune cells [71,72]. As expected because of the stimulatory effect on TLR2 and 4, upregulated genes within TLR-related pathways were observed. C-Jun, the encoded protein of JUN, combines with c-Fos to form the AP-1 early response transcription factor within the JNK pathway [73,74]. C-jun plays a role in cell cycle progression through the G1 phase and stimulates proliferation [75]. It also exerts an anti-apoptotic effect, by cooperating with NF-κB to prevent apoptosis induced by TNFα [75]. MAP2K3 is activated by mitogenic and environmental stress, and is regulated by NF- κB as a transcriptional cofactor [76]. It can be activated by insulin, and is necessary for the expression of glucose transporter molecules [77], indicating it also plays a role in metabolic processes. MAPK13 is a kinase in the p38 MAP kinase family, which can be activated by proinflammatory cytokines and cellular stress [78,79]. MAPK13 substrates include the transcription factor ATF2 and the microtubule dynamics regulator stathmin, and it is reported to play a role in differentiation, apoptosis, and inflammatory diseases [80,81]. Upregulation of these genes within TLR- related pathways demonstrates that cellulose not only exerts effects on genes which are directly related to innate immune reactivity but also impacts on genes involved in cell cycle regulation and metabolic processes of immune cells. Results from the electrical cell substrate impedance sensing experiments indicate that cellulose does not exert direct barrier stimulating or protective effects in this in vitro model. TLR2 is an important regulator of epithelial resistance, and some dietary fibers indeed show barrier protective effects mediated by TLR2 binding [23]. Plausibly, the low binding and activation of TLR2 by cellulose could explain this lack of effect. Despite these results, in vivo studies indicate that cellulose confers other important protective effects on intestinal health, such as inducing production of SCFAs by the microbiota [13], and the capacity to adsorb a range of dietary carcinogens [82]. Overall, these results show that cellulose is immunomodulating. The expression of several categories of molecules within NF-κB and TLR- signaling was stimulated by cellulose, but also several genes within these pathways were downregulated in their expression. The activating potential of cellulose could be due to TLR activation and downstream

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Figure 5. Ingenuity Pathway Analysis showing significantly up-, and down regulated molecules within NF-κB signaling pathways upon stimulation of human PBMCs with LPS, as compared to control. Upregulated genes are indicated in red, downregulated genes are indicated in green.

signaling events, however this could also be an explanation for upregulation of inhibitory molecules within this pathway, which may be part of a negative feedback signal. These results warrant further studies into possible effects of cellulose on TLR/NF-κB activation and regulation of expression in vivo, and also implicate that cellulose may not be an

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CHAPTER 4. CELLULOSE ALTERS THE EXPRESSION OF NF-κB RELATED GENES AND TLR-RELATED GENES IN HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS. adequate control substance in in vivo or in vitro dietary fiber studies [83- 86], due to the potential effects on immune function. Although further studies are required to assess the functional impacts of cellulose and root pulp supplementation on the immune system, the immunostimulatory property of cellulose can be an additional health promoting characteristic contributing to the potential application of root vegetable byproduct in the functional food industry.

Figure 6. Ingenuity Pathway Analysis showing significantly up-, and down regulated molecules within TLR signaling pathways upon stimulation of human PBMCs with LPS, as compared to control. Upregulated genes are indicated in red, downregulated genes are indicated in green.

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Figure 7. Relative TEER values and AUCs of T84 monolayers treated with cellulose and PMA. A) Relative TEER values as percentage of starting value. B) Area under the curve of TEER, calculated over a 24 h interval starting from the addition of PMA, shown as a percentage of the AUC of untreated control cells. Legends indicate concentrations of the applied cellulose in µg / mL, (n=6).

Table 3. Fold induction of up-, and downregulated genes in the NF-κB pathway by LPS.

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Table 3 continued.

Table 4. Fold induction of up-, and downregulated genes related to TLR signaling by LPS.

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Table 4. continued.

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CHAPTER 4. CELLULOSE ALTERS THE EXPRESSION OF NF-κB RELATED GENES AND TLR-RELATED GENES IN HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS.

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CHAPTER 5

The impact of lemon pectin characteristics on Tlr activation and t84 intestinal epithelial barrier function

Leonie M. Vogt1*, Neha M. Sahasrabudhe1, Uttara Ramasamy2, Diederick Meyer3, Gerdie Pullens4, Marijke M. Faas1,5, Koen Venema6, Henk A. Schols2, Paul de Vos1

1Immunoendocrinology, Division of Medical Biology, Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, the Netherlands 2Laboratory of Food Chemistry, Wageningen University, PO Box 17, 6700 AA Wageningen, the Netherlands 3Sensus B.V., Borchwerf 3, 4704 RG Roosendaal, the Netherlands 4Cosun Food Technology Centre, Oostelijke Havendijk 15, 4704 RA, Roosendaal, the Netherlands 5Department of Obstetrics and Gynaecology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, the Netherlands 6Beneficial Microbes Consultancy, Johan Karschstraat 3, 6709 TN Wageningen, the Netherland

Submitted to Journal of Functional Foods.

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Abstract

Scope Metadata analyses confirm that dietary fibers reduce risk and incidence of disease. This study aimed to investigate underlying structure-response relationships of different degree of polymerization (DP) and degree of methyl esterification (DM) pectins as immunomodulatory and epithelial barrier active dietary fibers. Methods and Results. 30DM, 56DM and 74DM lemon pectins were applied to test effects of degree of methyl esterification. Enzymatic digests of these pectins were applied to study the effects of chain length modification. NF-κB/AP-1 activation was measured using TLR and MyD88 reporter assays. The pectins demonstrated TLR dependent activation, and an increasing TLR activation with increasing DM. Pectins induced TLR2, and TLR4 activation, and enzymatic digestion of the polymers into oligomers abrogated their TLR activating potential. Barrier function of human intestinal epithelial cells was quantified by resistance measurements across T84 monolayers. 30DM and 74DM pectins induced a strong barrier protective effect, while 56DM pectins induced moderate protection of T84 TEER. Conclusion. Activation of immune cells by lemon pectins is TLR dependent through ligation of TLR2 and 4, and the intact polymer backbone is indispensable for activation. Degree of methyl esterification is a determining factor in activation potential and epithelial barrier protective effects, which is strongest for 30 DM and 74 DM lemon pectins.

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Introduction

5.1 Background An important category of health promoting nutritional compounds, which are under extensive study, is that of dietary fibers [1]. Data from meta- analyses confirm that adequate intake of dietary fibers reduces the risk and incidence of diseases such as type 2 diabetes, cardiovascular diseases, cancer [2-5], several gastrointestinal disorders, and obesity [6]. Although dietary fibers have been shown to have a collective health promoting effect, it is largely unknown which chemical properties of which individual fiber are responsible for which of the observed effects. Dietary fibers comprise a spectrum of different molecules from a broad range of plant species, and can also be produced by chemical or enzymatic synthesis such as polydextrose or galactooligosaccharides (GOS) [7,8]. Moreover, within one category of dietary fibers, the physiological responses can differ depending on backbone chain length [9,10] and other physicochemical properties such as the number and the type of side chains [11]. Pectins form a large family of dietary fiber molecules with a range of different structural properties. They are important structural components of plant cells consisting of linear 1,4-α-D-galacturonan (homogalacturonan) segments and highly branched rhamnogalacturonan segment [12]. Industrially extracted pectins are used by the food industry as gelling agent. When pectin has more than 50% of the acid units in the backbone methyl-esterified (degree of methyl esterification – DM), it is usually classified as "high methyl ester (HM) pectin" [13]. Saponification of pectins will reduce the degree of esterification and will yield "low methyl ester (LM) pectins" (DM<50) [14]. Examples of pectin structures with different DM are depicted in Figure 1. Next to the absolute amount of methyl esters present within the pectin molecule, also the distribution of methyl esters over the galacturonan backbone may determine the techno- functional properties [15,16]. As previously shown, the degree of methyl esterification as well as the chain length of pectin molecules are important factors in inducing different biological effects [17], including effects on the consumer’s immune system [18-26]. Up to now these effects on the host immune system were mainly attributed to beneficial effects of the dietary fibers on the consumers’ microbiota. However, recently we have shown that dietary fibers can also have direct effects on immune cells. Several prebiotic fibers

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CHAPTER 5. THE IMPACT OF LEMON PECTIN CHARACTERISTICS ON TLR ACTIVATION ANT T84 INTESTINAL EPITHELIAL BARRIER FUNCTION. have been shown to directly ligate sensors of the innate immune system, the so-called pattern recognition receptors (PRRs) [9,27,28]. Toll-like receptors (TLRs) are the best characterized PRRs and have been shown to be involved in signaling of dietary fibers [9,27,29,30]. We hypothesized that activation of TLRs may be one of the factors involved in the immunomodulatory properties of pectins. To address potential structure- function relationships we compared lemon pectins which can be obtained in defined chemical compositions. We investigated the role of chain length and degree of methyl esterification of pectins on TLRs. Also we studied the effects of pectin with different degree of methyl esterification on barrier function of human intestinal cells in vitro, as pectins might exert health effects by modulating the intestinal barrier [31,32]. To this end we applied a phorbol 12-myristate-13-acetate (PMA) damage model to T84 intestinal epithelial monolayers.

Methods

5.2 Investigational compounds, enzymatic digestion, and chemical characterization with high performance anion exchange chromatography and high pressure size exclusion chromatography The investigational compounds applied in this study were lemon pectins with different degrees of methyl esterification, i.e. 30% (30DM), 56% (56DM), and 74% (74DM), acquired from CP Kelco; Lille Skensved, Denmark (see also Figure 1). The pectins have been described in detail by Daas et al. (2000) with the codes C30, C55, and C74 respectively. These compounds were applied to investigate structure-function relationships for the degree of methyl esterification. In some experiments, the pectins were digested to oligomers to confirm that the biological effects are dependent on an intact backbone of the molecule or whether they can also be achieved by the oligomers. To this end, the pectin substrates (30DM, 56DM and 74DM; 500 mg) were dissolved in 50 mL of 50 mM ammonium acetate buffer, pH 5. The enzymes used were endopolygalacturonase (EC 3.2.1.15) from Kluyveromyces fragilis with an activity of 68.68 U/mL, 20.68 µL was added [33] (for DM 30 and DM55 pectin) and pectin lyase II (PL, EC 4.2.2.10), from Aspergillus niger, with an activity of 8.8 U/mL, 161.41 µL was added [34] for DM74 pectin. Enzyme incubations were performed for 10 h, rotating at 125 rpm at 30°C. Substrate blanks and enzyme blanks were prepared as controls. Following

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Figure 1. Schematic representation of different DM pectins. A) 30DM pectin. B) 56DM pectin. C) 74DM pectin. Methylated galacturonic-residues are indicated in grey, unmethylated in white.

incubations for 10 h, all samples were boiled for 5 min to inactivate the enzymes. Chemicals were from Sigma Aldrich Chemie B.V., Zwijndrecht, the Netherlands. Chemical characterization by high performance anion exchange chromatography (HPAEC) and high pressure size exclusion chromatography (HPSEC) of the untreated and treated pectins was performed as previously described [9]. Results of the chemical characterization are shown in Figure 2.

5.3 Cell culture T84 human intestinal epithelial cells were acquired from Sigma Aldrich Chemie B.V. Cells were cultured as previously described [35,36]. THP1 MD2-CD14, THP1 DefMyD, HEK-Blue hTLR2, and HEK-Blue hTLR4 reporter cell lines, Normocin antibiotic, and selection media were all acquired from InvivoGen, Toulouse, France. Reporter cells were cultured as previously described [9]. Briefly, the THP-1 cell lines were cultured in RPMI1640 supplemented with 10% heat inactivated FBS (60⁰C, 60 min), glucose (4.5 g/L), NaHCO3 (Boom B.V., Meppel, The Netherlands;1.5 g/L), HEPES (10

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CHAPTER 5. THE IMPACT OF LEMON PECTIN CHARACTERISTICS ON TLR ACTIVATION ANT T84 INTESTINAL EPITHELIAL BARRIER FUNCTION. mM), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 mg/mL), and sodium pyruvate (1 mM) all from Sigma-Aldrich Chemie B.V., and with Normocin (100 mg/mL, from InvivoGen). THP-1 cell lines were maintained at a concentration of 5*105 cells/mL. After culturing for 3 passages, THP1 cell lines were maintained in selection media by adding Zeocin (200 µg/mL) and G418 (250 µg/mL) to the THP1 MD2-CD14 cell culture medium, and by adding Zeocin (200 µg/mL) and HygroGold (100 µg/mL) to the THP1 DefMyD cell culture medium. HEK-Blue cells were cultured in DMEM (Life Technologies Europe B.V., Bleiswijk, the Netherlands), supplemented with glucose (4.5 g/L), L-glutamine (2 mM), 10% heat inactivated FBS, penicillin/streptomycin (50 U/mL and 50 mg/mL), and Normocin (100 mg/mL). HEK cells were grown to ca. 80% confluency. After culturing for 3 passages, the HEK cell lines were maintained in selection media by adding 1X HEK-Blue Selection to the culture medium.

5.4 Reporter cell assays To establish whether lemon pectin can activate PRRs, and more specifically TLRs, TLR reporter cell assays were performed according to the manufacturer’s protocol. Briefly, THP-1 MD2-CD14 cells expressing all TLRs were resuspended to a cell density of 1*106 cells/mL, 100 µL of this cell suspension per well was stimulated for 24 h (37⁰C, 5% CO2) with untreated and treated pectic compounds (described above), or 100 ng/mL ultrapure E.coli LPS (InvivoGen) as a positive control. Culture medium was incorporated in the assay as negative control. Subsequently 20 µL of supernatant of the stimulated reporter cells was incubated with 180 µL of

Quanti-blue reagent (InvivoGen; 45 min, 37⁰C, 5% CO2) and NF-κB/AP-1 was measured by the amount of excreted SEAP and Quanti-blue color conversion (OD) at 650 nm using a VersaMax microplate reader (Molecular Devices GmbH, Biberach an der Riss, Germany) and SoftMax Pro Data Acquisition & Analysis Software. To study whether activation of NF-κB/AP-1 in the THP-1 MD2- CD14 cells was fully TLR-dependent, THP-1 cells expressing all TLRs but with a non-functional TLR adapter MyD88 were applied. These cells were resuspended to a density of 2*106 cells/mL and the reporter assay was performed as described above for THP-1 MD2-CD14 cells, with the exception that the positive control for MyD88 independent stimulation of THP1-DefMyD cells was 25 µg/mL Tri-DAP (InvivoGen). According to recent literature reports, TLR2 and TLR4 appear to be two main TLRs by which dietary fibers can induce direct cellular activation

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[9,37-40]. Therefore, we next studied the activating capacity of the pectic compounds for activation of TLR2 and TLR4 in reporter cells. HEK-Blue hTLR2 reporter cells were resuspended to a cell density of 2.8*105 cells/mL, and HEK-Blue hTLR4 reporter cells were resuspended to a cell density of 1.4*105 cells/mL. 180 µL of cell suspension was stimulated for 24 h (37⁰C, 5% CO2) with 20 µL of the pectic compounds described above, or 20 µL of culture medium as negative control, or 20 µL of a suspension of 1*108 heat killed Listeria Monocytogenes cells per ml (InvivoGen) or 100 ng/mL ultrapure E.coli LPS as positive control. Analysis of NF-κB/AP-1 activation was performed as described for the THP-1 cells.

5.5 TEER of T84 human intestinal cells Epithelial resistance across differentiated monolayers of T84 intestinal cells was determined using gold-plated electrode chamber slides and Epithelial Cell Substrate Impedance Sensing as described previously [36] with some modifications. Briefly, multiple electrode gold-plated 96 well chamber slides (96W20idf, Applied Biophysics, IBIDI, München, Germany) were coated with 300 µL/well of a 0.2% L-cysteine (Sigma-Aldrich Chemie B.V.) solution in DMEM (Life Technologies Europe B.V.) for 30 min at room temperature. Wells were washed twice with DMEM, and coated overnight at room temperature with 300 µL/well of 1% PureColTM bovine tail collagen (Nutacon B.V., Leimuiden, the Netherlands) and 0.1% BSA (Sigma- Aldrich Chemie B.V.) in culture medium. Wells were then washed twice with culture medium and cells were seeded at a density of 2*105 cells per well in a final volume of 300 μL/well. Prior to stimulation, the cells were maintained in the wells for 14 days to reach a stable TEER, and medium was changed every other day. Resistance was measured continuously at multiple frequencies [41] upon placing the chamber slides in an ECIS incubator (Z-Theta model, Applied Biophysics, Troy, New York, USA). Measurements performed at 500 Hz were used to calculate the AUC, as relatively low frequencies are specifically representative for the tight junction mediated resistance [41]. To establish whether pectins exert protective effects, a damage model was applied based on T84 cell incubation with phorbol 12-myristate 13-acetate (PMA, 10nM, Sigma- Aldrich Chemie B.V.), which is a barrier disrupting agent [42]. T84 cells were incubated with DM30, DM56, and DM70 pectins for 24 h, followed by addition of PMA. Cells were maintained in this stimulation medium for at least 20 h, and this time frame of 20 h was subsequently used to calculate the AUC relative to untreated controls.

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5.6 Statistical analysis GraphPad Prism version 5.0 was used for statistical analysis of all reporter cell data and all data from the ECIS experiments. Significance levels were determined by Wilcoxon signed rank test and one-way ANOVA. Results are expressed as median and interquartile range. p-values < 0.05 are considered statistically significant and are denoted with *.

Results

5.7 Chemical characterization of lemon pectins The pectins DM30, DM56, and DM74 used in this study have a similar molecular weight (40-100 kDa) (Figure 2A-C). All three pectins had a rather random distribution of their methyl esters. To study the effect of size of the pectin molecules, the pectins were degraded by enzymes to oligomeric fragments. Digests were studied with HPSEC and HPAEC (Figures 2 A-D). The 30DM digest after treatment with polygalacturonase consists of saturated GalA oligomers of DP2-DP20, with the main fraction being DP2-DP6 (Figure 2A, 2D). The 56 DM polygalacturonase digest was less degraded by the enzyme but still consists of lower Mw fragments (Figure 2B, 2D). The digestion of 74DM pectin was performed by pectin lyase since polygalacturonase is hindered too much by methyl esters in the 74DM pectin. The HPSEC pattern in Figure 2C demonstrates a successful degradation to low Mw material. However, due to the working mechanism of the enzyme, mainly unsaturated GalA oligomers of DP2- DP14 are present in the digest (Figure 2D).

5.8 Activation of THP1MD2-CD14 cells by lemon pectin polymers is dependent on TLR activation To study whether lemon pectins can activate immune cells, culminating in NF-κB/AP-1 induction, DM56 pectin was co-incubated with THP1 MD2- CD14 cells equipped with a reporter gene. As shown in Figure 3A we observed that pectin activated the THP1 MD2-CD14 cells with a 4.4 fold- induction (± 0.19 interquartile range, p < 0.05) as compared to control. Next, we studied whether the induced NF-κB/AP-1 activation in the THP1 MD2-CD14 cells was mediated by MyD88, i.e. the central adapter molecule for TLR signaling. This was done by comparing the activation of the THP1 MD2-CD14 cells which express all TLRs in

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CHAPTER 5. THE IMPACT OF LEMON PECTIN CHARACTERISTICS ON TLR ACTIVATION ANT T84 INTESTINAL EPITHELIAL BARRIER FUNCTION. conjunction with MyD88 [43], with THP1 DefMyD cells which only express all TLRs, but a truncated, inactive form of MyD88 [9].

Figure 2 (continued). HPSEC elution patterns of the untreated pectin and of the corresponding enzyme digest, as a measure for their molecular weight distribution profiles in kDa for A) DM30 pectin; B) DM55 pectin; and C) DM74 pectin. D) HPAEC profiles of the pectin oligomers present in the enzyme digests of DM30, DM55, and DM74 pectins. GalA, galacturonic acid, uGalA, unsaturated galacturonic acid Subscript number indicates length of oligomer. Mw; Molecular weight, DM; Degree of Methyl esterification, DDM30; digested 30DM pectin, DDM56; digested 56DM pectin; DDM74; digested 74DM pectin.

As shown in Figure 3B, the pectin induced no activation of the MyD88 deficient cell line, indicating that the observed activation in the THP1 MD2-CD14 cell line was dependent on MyD88 signaling and thus TLR-mediated.

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Figure 2 (continued).

50 A 50 B 30 * 30 * 6 6

4 * 4

Fold induction Fold 2 2

compared to control to compared

0 0 56DM pectin control LPS 56DM pectin control Tri-DAP

Figure 3. Stimulation of MyD88 proficient-, and deficient TLR reporter cells with 56DM lemon pectin. Figure A shows the results of fully functional THP-1 MD2-CD14 cells expressing all human TLRs, and Figure B depicts the results for THP-1 reporter cells expressing all human TLRs but which are deficient in MyD88 signaling (THP1 DefMyD cells). Both cell lines were incubated with 1 mg/mL 56DM lemon pectin, culture medium as negative control, and 100 ng/mL LPS or 25 µg/mL Tri-DAP as positive controls respectively. Statistical significance was determined with a Wilcoxon signed rank test, and activation compared to control is indicated with an asterisk. Median and interquartile range of activation is plotted as fold induction as compared to control (n = 8).

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5.9 TLR induced NF-κB/AP-1 activation by lemon pectin is dependent on the degree of methyl esterification Next the effect of the degree of methyl esterification on immunological properties of pectin was investigated by comparing the TLR-mediated NF- κB/AP-1 activation in THP1 MD2-CD14 cells after exposure to pectin polymers with a DM value of 30, 56, and 74. As shown in Figure 4A, the higher the degree of methyl esterification of the pectin polymers, the higher the TLR-mediated NF- κB/AP-1 activation in THP1 MD2-CD14 cells. The 30DM pectin polymers induced a 3-fold induction of activation compared to control (± 0.86 interquartile range, p < 0.05), the 56DM pectin polymers induced a 4.4- fold induction of activation (± 0.19 interquartile range, p < 0.05) and 74DM pectin polymers induced the strongest activation, i.e. a 5-fold induction as compared to control (± 0.92 interquartile range, p < 0.05). Comparison between the different DM fractions indicated that 56DM and 74DM pectins induced significantly stronger activation as compared to 30DM pectins and 74DM pectins trended to induce stronger activation compared to 56DM pectins (p < 0.1). These results show that the structural property of degree of methyl esterification in pectins is an important feature determining the TLR-activating capacity of lemon pectins.

5.10 TLR dependent NF-κB/AP-1 activation by lemon pectin requires an intact backbone To determine whether the TLR-mediated NF-κB/AP-1 activation by lemon pectin is dependent on its molecular weight or can also be achieved by oligomers of pectins, the degree of polymerization was enzymatically reduced to pectin oligomers. Reduction of the polymer length resulted in loss of TLR activation (Figure 4B). The oligomers did not have the same effect as the full pectins with an intact backbone, suggesting that backbone chain length is an important factor for the activating potential of these lemon pectins.

5.11 Different DM pectins show different activation patterns of TLR2 and TLR4 Next it was studied whether TLR2 and TLR4 are involved in the activation. TLR2 and TRL4 can recognize polymeric carbohydrates [44-46] and are both implicated in the recognition of dietary fibers [9,37-40]. Therefore it was investigated whether the NF-κB/AP-1 activation by pectins is TLR2 or TLR4 dependent. HEK-Blue TLR reporter cells with either human TLR2 or TLR4 constructs were stimulated with concentration series of pectin

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50 50 A B 30 * 30 6 * 6

4 4

Fold induction Fold 2 2

compared to control comparedto

0 0 30DM 56DM 74DM control LPS 30DM 56DM 74DM control LPS

PECTIN POLYMERS PECTIN DIGESTS

Figure 4. Activation of THP1 MD2-CD14 cells with polymeric pectins and digests of pectins of different DM (1 mg/mL). Figure A depicts activation of the cell line upon stimulation with different DM pectin polymers. Figure B shows the effect of oligomeric digests of the different DM fractions on THP1 activation. Statistical significance was determined with a one-way ANOVA and differences between the different DM fractions are indicated with an asterisk. Median and interquartile range of activation is plotted as fold induction as compared to control (n = 8).

20 A * 20 B * 10 10

8 8 6 6 * 4 4 * 2 2 * 0 0 30DM 56DM 74DM HKLM control 30DM 56DM 74DM HKLM control

100 µg/mL 200 µg/mL

20 C * 20 D * 10 10 *

8 8 6 * 6 4 4

2 * 2 * * Fold induction compared to control to compared induction Fold 0 0 30DM 56DM 74DM HKLM control 30DM 56DM 74DM HKLM control

400 µg/mL 1000 µg/mL Treatment and concentration (microgram/ml)

Figure 5. Stimulation of HEK-Blue hTLR2 cells with a concentration series of different DM pectin polymers. A) 100 µg/ml dose. B) 200 µg/ml dose. C) 400 µg/ml dose. D) 1000 µg/ml dose. Differences compared to control were assessed with Wilcoxon signed rank test, and are indicated by an asterisk. Median and interquartile range of activation is plotted as fold induction as compared to controls (n = 8).

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CHAPTER 5. THE IMPACT OF LEMON PECTIN CHARACTERISTICS ON TLR ACTIVATION ANT T84 INTESTINAL EPITHELIAL BARRIER FUNCTION. polymers. As depicted in Figure 5A-D, DM30 pectins only activated TLR2 at the concentrations 200 and 400 µg/mL, and DM56 pectins only activated TLR2 at the concentrations of 400 and 1000 µg/mL. In addition, both DM30 and DM56 pectins only induced moderate activation. However, DM74 pectins induced a relatively strong, and dose dependent TLR2 activation over the whole concentration range tested, reaching a ca. 9-fold induction of activation at the highest concentration (1000 μg/mL, p < 0.05). Figure 6 illustrates that low dosed pectins of different DM, all induced a ca. 4-fold induction of TLR4 activation, and that upon increase of the dose, only a slight increase in activation was observed as compared to the lowest dose. A subtle DM effect appears to be present suggesting that with increasing DM, pectins induce increased activation, however upon testing for multiple parameters with one-way ANOVA, these differences proved not significant. Taken together, these results demonstrate that high DM pectins can dose-dependently activate TLR2, and can also activate TLR4 in a less pronounced manner, and that lower DM pectins can activate both TLR2 and TLR4, but TLR2 activation was much less pronounced as compared to high DM pectins.

5.12 Effect of different DM pectins on TEER of T84 intestinal epithelial cells To study whether lemon pectins protect T84 cells against PMA-induced loss of TEER and whether the DM value is important in TEER modulation, T84 cells were incubated with lemon pectin polymers with a degree of methyl esterification of 30, 56, and 74, for 24 h. Then PMA was added and the AUC for a 20-h time period after PMA addition was plotted for the different pectin treatments, as a percentage of the AUC of untreated controls, which was set to 100% (Figure 7). PMA treatment caused a decrease in T84 TEER resulting in an AUC of 54.3 ± 3.4% (p < 0.01) as compared to control, confirming that damage was induced to the epithelial barrier. Upon 24 h of preincubation of T84 cells with 100 µg/L DM30, DM56, and DM74 polymers, all compounds conferred protection against PMA-induced loss of resistance with TEER AUC values reaching 93.9 ± 24.6% (p < 0.05), 59.9 ± 2.2% (p < 0.05), and 77.6 ± 15.2% (p < 0.05) respectively, as compared to control. These results suggest that 30DM and 74DM pectins confer considerable protection on T84 cells against a PMA-induced decrease in TEER, and that 56DM pectins exert moderate barrier protective properties.

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Figure 6. Stimulation of HEK-Blue hTLR4 cells with a concentration series of different DM pectin polymers. A) 100 µg/ml dose. B) 200 µg/ml dose. C) 400 µg/ml dose. D) 1000 µg/ml dose. Differences compared to control were assessed with Wilcoxon signed rank test, and are indicated by an asterisk. Median and interquartile range of activation is plotted as fold induction as compared to controls (n = 8).

Figure 7. Barrier function of T84 human intestinal epithelial cells represented by AUCs of TEER values in time. Statistical significance levels compared to PMA were determined with a Wilcoxon signed rank test, and differences are indicated by an asterisk. AUCs are plotted as percentage of control, which was set to 100% (n = 3).

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Discussion

The aim of this study was to investigate the structure-function relationship of lemon pectins by studying the effects of DP and DM on TLR-mediated cell activation, and on the barrier function of human intestinal epithelial cells. Since several dietary fibers are ligands for innate immune receptors [9,37-40], we hypothesized that pectins could also be one of the dietary fiber types capable of activating TLRs, and that this would be one of the mechanisms by which pectins exert their immunomodulatory effects. Upon stimulating a THP1 reporter cell line, which expresses all TLRs, we observed that lemon pectins activate these human monocytic cells. Neither of the pectin compounds activated the THP1 DefMyD cells, confirming that the observed activity was mediated via TLRs. TLRs mediated immune activation can also explain the in vivo described immune effects of pectin. Immune activating properties of lemon pectin have been reported in animal studies which showed that supplementation prevented the induction of oral tolerance to OVA in rats, which was preceded by enhanced protein antigen penetration to the blood and activation of macrophages [47]. Moreover, in that study, lemon pectin enhanced IFNγ and TNFα production by peritoneal macrophages. In a study by Suh et al. [48], a lemon derived pectic-type polysaccharide was orally administered to mice, causing increased secretion of GM-CSF and IL- 6 from Peyer’s patches, indicating immune cell activation. Some studies report immunomodulatory effects of the oligomer fractions which remain after enzymatic digestion of pectins [11]. Although these oligomers often only induced immunomodulation in combination with galactooligosaccharides and fructooligosaccharides [49,50] we decided to test such digests for their TLR activating capacities. By incubating THP1 MD2-CD14 cells with untreated and digested pectin polymers we observed that the intact polymers induced NF-κB/AP-1 activation, but the digested fractions showed no activating capacity. The absence of activation by the digests indicates that the intact polymeric lemon pectin molecules are required for activation, and that the oligomers derived after digestion had lost this property. This suggests that the direct immunostimulatory effects of pectins will probably occur in the small intestine, as the pectins are likely to be digested by bacteria in the colon. The importance of the backbone of pectins has been thoroughly reviewed by Popov et al. [11]. In a range of vegetables, the types of pectins were characterized and the effects on immune cells were evaluated. Important

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CHAPTER 5. THE IMPACT OF LEMON PECTIN CHARACTERISTICS ON TLR ACTIVATION ANT T84 INTESTINAL EPITHELIAL BARRIER FUNCTION. conclusions from the different types of pectins of different origins were that structural differences within pectin subtypes can induce different effects on immune cells. More specifically for lemon type pectins, the aforementioned study by Suh et al [48] also confirmed that the intact backbone was necessary for immunostimulatory effects in Peyer’s patches cells, as the hydrolysate of the pectin lost its activating potential. Our experiments subscribe that the intact backbone of lemon pectins is an important structural and functional feature, as it determines the TLR- activating capacity. Next, when comparing the untreated polymers with different DMs for their activating potential, we observed that higher DMs were associated with higher activation of THP1 MD2-CD14 cells. This activating effect for high DM lemon pectins, and inhibitory effect on immune cells was previously reported by Popov et al., [51,52] and Chen et al. [51,52]. In the latter study, different DM pectins were studied for their LPS-inhibiting effect on macrophages, and the highest DM pectin demonstrated the greatest inhibition. As the pectins were not studied without the presence of LPS, the intrinsic activation capacity of the pectins themselves was not determined in that study. Nevertheless, the inhibitory mechanism suggests that there may be a competitive interaction from the high DM pectins with TLR4. From our results it can be concluded that besides DP, also DM is a structural feature, which plays a role in the immunomodulatory potential and outcome upon immune cell contact with this particular type of pectins. Subsequently, we studied whether the TLR-mediated activation could be attributed to TLR2 and/or TLR4 by applying the pectin compounds to HEK-Blue hTLR2 and HEK-Blue TLR4 cells. TLR2 and TLR4 are the TLRs which have so far been identified as innate receptors important in carbohydrate fiber recognition. In addition, for apple pectin an LPS- inhibiting interaction with TLR4 has been reported [53]. As the digests showed no TLR-activating properties in the THP1 cells, only the intact polymeric pectins of different DMs were applied to the HEK reporter cells. We observed that only the higher DM pectins were capable of TLR2 activation, but that all pectins, already at low doses, readily activated TLR4. These results suggest that the activating potential of lemon pectins as observed in the THP1 cells could be attributed to both TLR2, and TLR4 activation. Several mechanisms for TLR-mediated immune modulation by pectins can be suggested. As a TLR4 ligand, pectin could bind to TLR4 and in this way cause competitive inhibition of LPS-mediated activation. Although TLR4 ligation is best known for its stimulating effect on immune

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CHAPTER 5. THE IMPACT OF LEMON PECTIN CHARACTERISTICS ON TLR ACTIVATION ANT T84 INTESTINAL EPITHELIAL BARRIER FUNCTION. reactions, and TLR2 is involved in defense against several categories of pathogens, TLR2 ligation can also induce anti-inflammatory responses, depending on its co-signaling molecules and also depending on tissue type or even type of organism [36,54-56]. In addition, TLRs have been shown to interact strongly with each other’s signaling pathways [57,58] so the different outcomes in immune modulation by different DM pectins could also be related to the ratio between TLR2/TLR4 activation. TLR2 serves as an an important barrier regulatory receptor [56], and TLR2 ligation by certain types of fibers can induce beneficial effects on intestinal epithelial cells [36]. Therefore we investigated whether incubation of T84 intestinal epithelial cells with pectins could protect the barrier function against the barrier damaging agent, PMA. In previous studies in our group, the TLR2-activating capacity of the dietary fibers short chain inulin-type fructans exerted preventive effects against barrier loss in human intestinal epithelial cells [36], and TLR2 is an important innate immune receptor with a crucial role in intestinal barrier regulation [56]. When we applied PMA to the T84 cells a decrease in TEER was observed, and by preincubating with pectins, this decrease was ameliorated, confirming a protective effect of pectins on epithelial barrier function. In two studies, bacterial infection or NSAID treatment were applied to broiler chicks and cats respectively, as challenge to the intestinal barrier [59,60]. In these studies protective effects of pectin supplementation were also observed regarding intestinal integrity and barrier function [59,60], suggesting that pectin-mediated protection of the intestinal barrier is not restricted to one damage model and can be beneficial against several toxins or pathogens. In conclusion, DP as well as DM are physicochemical properties of lemon pectin which can determine their immunostimulatory properties and therefore may be relevant in the use of pectins for improvement of immune status. The implications of our results for functional food applications are that pectin fibers are promising immunomodulatory agents, but that studies into the physiological and more specifically the immunomodulatory effects of pectins should be meticulously designed. The origin, the extraction method, and the full chemical characteristics such as molecular weight and level and distribution of methyl esters may explain deviating observations for the immunomodulatory effects of pectins and should be included in publications in the future. Ultimately, this will facilitate side-by-side comparisons of reported immunomodulatory effects of all the different types of pectins available, providing an opportunity to develop tailored pectin fortified foods, with

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CHAPTER 5. THE IMPACT OF LEMON PECTIN CHARACTERISTICS ON TLR ACTIVATION ANT T84 INTESTINAL EPITHELIAL BARRIER FUNCTION. well described effects supported by knowledge of the underlying physiological responses.

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20. Vos AP, Haarman M, van Ginkel JW, Knol J, Garssen J, Stahl B, et al. Dietary supplementation of neutral and acidic oligosaccharides enhances Th1-dependent vaccination responses in mice. Pediatr Allergy Immunol. 2007;18: 304-312. 21. Popov SV, Popova GY, Nikolaeva SY, Golovchenko VV, Ovodova RG. Immunostimulating activity of pectic polysaccharide from bergenia crassifolia (L.) fritsch. Phytother Res. 2005;19: 1052-1056. 22. Sakurai MH, Matsumoto T, Kiyohara H, Yamada H. B-cell proliferation activity of pectic polysaccharide from a medicinal herb, the roots of bupleurum falcatum L. and its structural requirement. Immunology. 1999;97: 540-547. 23. Kiyohara H, Matsumoto T, Takemoto N, Kawamura H, Komatsu Y, Yamada H. Effect of oral administration of a pectic polysaccharide fraction from a kampo (japanese herbal) medicine "juzen-taiho-to" on antibody response of mice. Planta Med. 1995;61: 429-434. 24. Nergard CS, Matsumoto T, Inngjerdingen M, Inngjerdingen K, Hokputsa S, Harding SE, et al. Structural and immunological studies of a pectin and a pectic arabinogalactan from Vernonia kotschyana sch. Bip. ex walp. (asteraceae). Carbohydr Res. 2005;340: 115-130. 25. Michaelsen TE, Gilje A, Samuelsen AB, Hogasen K, Paulsen BS. Interaction between human complement and a pectin type polysaccharide fraction, PMII, from the leaves of Plantago major L. Scand J Immunol. 2000;52: 483-490. 26. Popov SV, Golovchenko VV, Ovodova RG, Smirnov VV, Khramova DS, Popova GY, et al. Characterisation of the oral adjuvant effect of lemnan, a pectic polysaccharide of Lemna minor L. Vaccine. 2006;24: 5413-5419. 27. Brown GD, Taylor PR, Reid DM, Willment JA, Williams DL, Martinez-Pomares L, et al. Dectin-1 is a major beta-glucan receptor on macrophages. J Exp Med. 2002;196: 407- 412. 28. de Kivit S, Kraneveld AD, Garssen J, Willemsen LE. Glycan recognition at the interface of the intestinal immune system: Target for immune modulation via dietary components. Eur J Pharmacol. 2011;668 Suppl 1:S124-32. 29. Underhill DM, Ozinsky A, Hajjar AM, Stevens A, Wilson CB, Bassetti M, et al. The toll- like receptor 2 is recruited to macrophage phagosomes and discriminates between pathogens. Nature. 1999;401: 811-815. 30. Underhill DM, Rossnagle E, Lowell CA, Simmons RM. Dectin-1 activates syk tyrosine kinase in a dynamic subset of macrophages for reactive oxygen production. Blood. 2005;106: 2543-2550. 31. Mao Y, Kasravi B, Nobaek S, Wang LQ, Adawi D, Roos G, et al. Pectin-supplemented enteral diet reduces the severity of methotrexate induced enterocolitis in rats. Scand J Gastroenterol. 1996;31: 558-567. 32. Rabbani GH, Teka T, Saha SK, Zaman B, Majid N, Khatun M, et al. Green banana and pectin improve small intestinal permeability and reduce fluid loss in Bangladeshi children with persistent diarrhea. Dig Dis Sci. 2004;49: 475-484. 33. Sakai T, Okushama M, Yoshitake S. Purification, crystallization and some properties of endopolygalacturonase from Kluyveromyces fragilis. Agric Biol Chem. 1984;48: 1951- 1961. 34. Kester HCM, Visser J. Purification and characterization of pectin lyase B, a novel pectinolytic enzyme from Aspergillus niger. FEMS Microbiol Lett. 1994;120: 63-67. 35. Marcon MA, McCool D, Forstner J, Forstner G. Inhibition of mucin secretion in a colonic adenocarcinoma cell line by DIDS and potassium channel blockers. Biochim Biophys Acta. 1990;1052: 17-23. 36. Vogt LM, Meyer D, Pullens G, Faas MM, Venema K, Ramasamy U, et al. Toll-like receptor 2 activation by β2→1-fructans protects barrier function of T84 human

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intestinal epithelial cells in a chain length dependent manner. J Nutr. 2014;144: 1002-1003-1008. 37. Tsai CC, Lin CR, Tsai HY, Chen CJ, Li WT, Yu HM, et al. The immunologically active oligosaccharides isolated from wheatgrass modulate monocytes via toll-like receptor-2 signaling. J Biol Chem. 2013;288: 17689-17697. 38. Lin CC, Chen HH, Chen YK, Chang HC, Lin PY, Pan IH, et al. Rice bran feruloylated oligosaccharides activate dendritic cells via toll-like receptor 2 and 4 signaling. Molecules. 2014;19: 5325-5347. 39. Kim MY, Muto J, Gallo RL. Hyaluronic acid oligosaccharides suppress TLR3-dependent cytokine expression in a TLR4-dependent manner. PLoS One. 2013;8: e72421. 40. Capitan-Canadas F, Ortega-Gonzalez M, Guadix E, Zarzuelo A, Suarez MD, de Medina FS, et al. Prebiotic oligosaccharides directly modulate proinflammatory cytokine production in monocytes via activation of TLR4. Mol Nutr Food Res. 2014;58: 1098- 1110. 41. Lo CM, Keese CR, Giaever I. Impedance analysis of MDCK cells measured by electric cell-substrate impedance sensing. Biophys J. 1995;69: 2800-2807. 42. Tai YH, Flick J, Levine SA, Madara JL, Sharp GW, Donowitz M. Regulation of tight junction resistance in T84 monolayers by elevation in intracellular Ca2+: A protein kinase C effect. J Membr Biol. 1996;149: 71-79. 43. Zarember KA, Godowski PJ. Tissue expression of human toll-like receptors and differential regulation of toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol. 2002;168: 554-561. 44. Oliveira-Nascimento L, Massari P, Wetzler LM. The role of TLR2 in infection and immunity. Front Immunol. 2012;3: eCollection 2012. 45. Sato M, Sano H, Iwaki D, Kudo K, Konishi M, Takahashi H, et al. Direct binding of toll-like receptor 2 to zymosan, and zymosan-induced NF-kappa B activation and TNF-alpha secretion are down-regulated by lung collectin surfactant protein A. J Immunol. 2003;171: 417-425. 46. Park BS, Song DH, Kim HM, Choi BS, Lee H, Lee JO. The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. Nature. 2009;458: 1191- 1195. 47. Khramova DS, Popov SV, Golovchenko VV, Vityazev FV, Paderin NM, Ovodov YS. Abrogation of the oral tolerance to ovalbumin in mice by citrus pectin. Nutrition. 2009;25: 226-232. 48. Suh HJ, Yang HS, Ra KS, Noh DO, Kwon KH, Hwang JH, et al. Peyer's patch-mediated intestinal immune system modulating activity of pectic-type polysaccharide from peel of citrus unshiu. Food Chem. 2013;138: 1079-1086. 49. Vos AP, van Esch BC, Stahl B, M'Rabet L, Folkerts G, Nijkamp FP, et al. Dietary supplementation with specific oligosaccharide mixtures decreases parameters of allergic asthma in mice. Int Immunopharmacol. 2007;7: 1582-1587. 50. Kerperien J, Jeurink PV, Wehkamp T, van der Veer A, van de Kant HJ, Hofman GA, et al. Non-digestible oligosaccharides modulate intestinal immune activation and suppress cow's milk allergic symptoms. Pediatr Allergy Immunol. 2014;25: 747-748-754. 51. Popov S, Markov P, Popova G, Nikitina I, Efimova L, Ovodov YS. Anti-inflammatory activity of low and high methoxylated citrus pectins. Biomedicine and Preventive Nutrition. 2013;3: 59-60-63. 52. Chen CH, Sheu MT, Chen TF, Wang YC, Hou WC, Liu DZ, et al. Suppression of endotoxin- induced proinflammatory responses by citrus pectin through blocking LPS signaling pathways. Biochem Pharmacol. 2006;72: 1001-1009.

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53. Liu L, Li YH, Niu YB, Sun Y, Guo ZJ, Li Q, et al. An apple oligogalactan prevents against inflammation and carcinogenesis by targeting LPS/TLR4/NF-kappaB pathway in a mouse model of colitis-associated colon cancer. Carcinogenesis. 2010;31: 1822-1832. 54. Cario E, Gerken G, Podolsky DK. Toll-like receptor 2 enhances ZO-1-associated intestinal epithelial barrier integrity via protein kinase C. Gastroenterol. 2004;127: 224-238. 55. Cario E, Gerken G, Podolsky DK. Toll-like receptor 2 controls mucosal inflammation by regulating epithelial barrier function. Gastroenterol. 2007;132: 1359-1374. 56. Cario E. Barrier-protective function of intestinal epithelial toll-like receptor 2. Mucosal Immunol. 2008;1 Suppl 1: S62-6. 57. van Bergenhenegouwen J, Plantinga TS, Joosten LA, Netea MG, Folkerts G, Kraneveld AD, et al. TLR2 & co: A critical analysis of the complex interactions between TLR2 and coreceptors. J Leukoc Biol. 2013;94: 885-902. 58. Trinchieri G, Sher A. Cooperation of toll-like receptor signals in innate immune defence. Nat Rev Immunol. 2007;7: 179-190. 59. Wils-Plotz EL, Jenkins MC, Dilger RN. Modulation of the intestinal environment, innate immune response, and barrier function by dietary threonine and purified fiber during a coccidiosis challenge in broiler chicks. Poult Sci. 2013;92: 735-745. 60. Satoh H, Hara T, Murakawa D, Matsuura M, Takata K. Soluble dietary fiber protects against nonsteroidal anti-inflammatory drug-induced damage to the small intestine in cats. Dig Dis Sci. 2010;55: 1264-1271.

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Long chain inulin-type fructanS BUT NOT SHORT CHAIN inulin-type fructanS enhance hepatitis b vaccination response in young adults

Leonie M. Vogt1, Marijke M. Faas1,2, Paul de Vos1

1Immunoendocrinology, Division of Medical Biology, Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, the Netherlands 2Department of Obstetrics and Gynaecology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700 RB Groningen, the Netherlands

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Objective. Inulin-type fructans have shown immune modulation in human cells, cell lines, and in supplemented experimental animals, but this has not been confirmed in healthy, immunocompetent human populations so far. Enhancing vaccination efficacy by supplementing with nutritional compounds is an accepted method for studying nutritional immune effects. This study was aimed to analyze the vaccine potentiating effect of inulin-type fructans, and to study whether their degree of polymerization is a determinant in the strength of the immune response. By analyzing peripheral blood lymphocyte subsets, concomitant changes in these populations may give insight in underlying mechanisms of a stimulated immune response. Design. In this randomized double-blind placebo-controlled supplementation study, 40 healthy volunteers aged 18-29 (17 males, 23 females), were supplemented for 14 days with inulin-type fructans of DP10-60 (long chain), or inulin-type fructans of DP2-25 (short chain), or fructose as placebo (n=13, 13, 14 per group, 8 g in single dose per day). On day 7, all volunteers were vaccinated against hepatitis B (Engerix-B). Blood samples were collected at day 0 (basal samples), 7, 14, 21, and 35. Anti- HBsAg titer at day 0, 14, 21, and 35 was analyzed with an Abbot Architect immunoanalyzer. At all five time points, the percentages of different B cell, T cell, NK cell, and NKT cell populations within the total lymphocyte population and subsets within populations were analyzed using multi- parameter flow cytometry. Results. DP10-60 fructans stimulated the vaccine-specific antibody response at day 35. This was not observed with DP2-25 fructan. In addition, two responders were identified in the DP10-60 fructan group vs. no responders in either the DP2-25 fructan group or the placebo group. In the placebo group, but not in the fructan supplemented groups, at day 21, the percentage of IgM+ non-class switched memory B cells was increased as compared to basal samples. In the DP10-60 group and the placebo group at day 14 and 21, the percentage of transitional B cells was increased compared to basal samples, but not in the DP2-25 group. The percentage of CD161+ NK cells was increased at day 21 and day 35 for each supplement. In the DP10-60 group, the percentage of TBET+ Th1 cells was increased at day 35 compared to basal samples. The increase in CD45ROhi cells as percentage of CTLs was not significant in the placebo group, and occurred earlier in time in the DP10-60 group (day 14)

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CHAPTER 6. LONG CHAIN INULIN-TYPE FRUCTANS BUT NOT SHORT CHAIN INULIN-TYPE FRUCTANS ENHANCE HEPATITIS B VACCINATION RESPONSE IN YOUNG ADULTS. compared to the DP2-25 group (day 35). Finally, the percentage of CD45ROhi cells in the Th population was increased at day 14 and 35 compared to basal samples in the placebo group but was not significantly increased in the fructan supplemented groups. Conclusion. This study demonstrates clear structure-effector relationships for dietary fibers in immune responses in humans. In addition, it shows that using less efficacious vaccination protocols to prove efficacy of food components on immunity may be a more feasible approach than using efficacious vaccination protocols. Finally, it is demonstrates that orally taken bioactive ingredients can influence systemic responses against pathogens during vaccination.

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Introduction

6.1 Background Dietary fibers are considered an essential part of healthy nutrition. The health benefits of sufficient fiber intake comprise prevention of colorectal cancer 1, type 2 diabetes 2, cardiovascular disease 3, reduced risk of hyperlipidemia, hypercholesterolemia and hyperglycemia 4 5, and regulation of bowel habit 6-10. Because the role of the immune system in these protective effects is not completely clear, there is a call for evidence of immune modulation by ingestion of dietary fibers in human studies, and for structure–function studies 11 12. Inulin-type fructans are prebiotic dietary fibers with many health benefits. They are oligomers and polymers of fructose subunits, and often terminate in a glucose molecule 13. Besides the established effects on gut health and metabolism 11 12 14, evidence for immunostimulatory effects of inulin-type fructan consumption is accumulating. Results from ex vivo and in vitro experiments in human cells, cell lines, and from animal studies, support the notion that inulin-type fructans exert immune modulating effects. For an extensive overview of these features the reader is referred to a collection of literature reviews 15-19 . The underlying mechanisms for immune modulation by inulin-type fructans have been attributed to the selective stimulation of beneficial bacteria in the intestine, and their SCFA fermentation products 17. In addition, previous results from our group support the notion that these fibers can also exert direct effects on immune cells, by activating Toll-like receptors (TLRs) on monocytic cells, and inducing cytokine production in human peripheral blood mononuclear cells (PBMCs) upon in vitro stimulation 20. The degree of polymerization (DP, or chain length) of inulin-type fructans ranges between 2 and 60, and commercially available inulin-type fructan powders often contain mixtures of different DP fructans. Interestingly, in our studies the DP of inulin-type fructans proved to be an important determinant in skewing PBMC cytokine profiles; short chain enriched inulin-type fructans (DP2-25) induced an anti-inflammatory IL-10/IL-12 ratio, whereas the long chain enriched inulin-type fructans (DP10-60) produced a more proinflammatory, or immunostimulatory IL-10/IL-12 ratio. In addition, TLR2, which was dose dependently activated by the fructans, demonstrated increased activation with increasing average fructan DP. Based on these differences in in vitro direct effects on immune cells, we selected the DP2-25 fructans and DP10-60 fructans as supplements to

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CHAPTER 6. LONG CHAIN INULIN-TYPE FRUCTANS BUT NOT SHORT CHAIN INULIN-TYPE FRUCTANS ENHANCE HEPATITIS B VACCINATION RESPONSE IN YOUNG ADULTS. study in vivo effects in a human trial. So far, inulin-type fructan supplementation trials which were aimed at boosting the human immune system, commonly target immunocompromised populations such as infants and elderly. We hypothesize that inulin-type fructan supplementation can also be beneficial for healthy immunocompetent populations. A means to study immune modulation is by studying the effects of inulin-type fructan supplementation on building immunity against pathogens such as during an infection or during vaccination, where an immune reaction is evoked without inducing disease. These types of studies are also recommended by regulatory agencies such as the European Food Safety Authority (EFSA) to prove immune efficacy of food components 21. Up to now, human vaccine efficacy studies with oligo-, or polysaccharide food supplements have mainly been done with efficacious vaccination protocols such as measles 22, tetanus 23, influenza 23 24, or pneumococcal 24 vaccination. Demonstrating efficacy of a food component in these protocols requires high numbers of volunteers, and effects will always be modest. Vaccines which by themselves do not induce an immediate strong antibody response are to our opinion a more attractive option for application in nutritional immunity studies. Hepatitis B vaccination belongs to this category. Common vaccination programs use hepatitis B surface antigen (HBsAg) to immunize subjects, and require booster injections to reach protective antibody titers (i.e. above 100 IU/ml) 25. We hypothesize that by supplementing young adults with inulin- type fructans from 7 days before, until 7 days after the first injection of an HBsAg vaccination program, the antibody response against the vaccine will be improved compared to placebo supplemented participants. This could be represented by an earlier onset of titer development, an increased titer response on the final measuring time point, and/or the presence and number of ‘responders’, characterized as subjects with titers equal to or above 10 IU/mL. Due to the previous results in our group mentioned above, we expect that DP10-60 fructans will stimulate the immune system and enhance the vaccine-induced response as compared to DP2-25 fructans and placebo. Besides the vaccination efficacy, peripheral blood lymphocyte subsets of the supplemented subjects were studied using multi-parameter flow cytometry. Concomitant changes in these populations may give an indication of underlying mechanisms of a stimulated immune response and highlight possible differences induced in lymphocyte populations, related to difference in DP of fructan supplements.

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Methods

6.2 Investigational compounds Inulin from chicory is a polydisperse mixture of linear fructan oligomers and polymers coupled by means of β(2-1) bonds, and mostly with a terminal glucose unit. The number of fructose units in the chain (degree of polymerization, or DP) can vary naturally between 2 and 60. Supplement A (Frutafit®TEX! Sensus, Roosendaal, the Netherlands), is a natural powdered food ingredient based on chicory inulin, and known for its texturizing properties. The DP ranges between 10 and 60. Supplement B (Frutafit®CLR Sensus), is a powdered fructo-oligosaccharide (FOS) produced by partial hydrolysis of chicory inulin. Frutafit®CLR is a highly soluble food ingredient with a DP ranging mainly between 2 and 25. Supplement C consists of fructose, which is a powdered carbohydrate, and serves as placebo because it consists of the monomer building blocks of fructans and does not have β(2-1) bonds. The DP profiles of supplement A and B are depicted in Figure 1.

Figure 1. DP profiles of supplement A and supplement B.

Figure 2. Schematic overview of experimental procedures in time.

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6.3 Volunteers and interventions This study was approved by the ethical board of the University Medical Center Groningen, Medisch Ethische Toetsingscommissie University Medical Center Groningen, and documented in the approved application METC_097. It has been registered in the national Dutch trial register, Nederlands Trial Register (NTR41644). Written informed consent was obtained from all participants, and data was analyzed and presented anonymously. Hepatitis B vaccination and blood sampling was conducted within the University Medical Center Groningen, in the Netherlands. All clinical investigation was conducted according to the principles expressed in the Declaration of Helsinki. A randomized double-blind placebo controlled human dietary intervention trial was designed to study the effect of inulin-type fructans on vaccination efficacy and peripheral blood lymphocyte populations. To study whether the degree of polymerization (DP) of inulin-type fructans can influence whether different types of immune reactions are stimulated, three groups were included in the study. Healthy volunteers without a history of gastrointestinal symptoms and free of medication, aged 18-29 (17 males, 23 females), were supplemented for 14 days with inulin-type fructans of DP10-60, or with inulin-type fructans of DP2-25, or fructose (Sigma-Aldrich, the Netherlands) as placebo (n=13, 13, and 14 per group respectively, 8g/d in one dose per day), and vaccinated against hepatitis B (Engerix-B, GlaxoSmithKline Biologicals s.a, Belgium) on day 7. The volunteers consumed their habitual diet and filled out a nutrition diary for the 35 days of the study. Blood samples were collected at day 0, 7, 14, 21, and 35, see Figure 2 for a schematic overview of experimental interventions.

6.4 Peripheral blood lymphocyte isolation and multi-parameter flow cytometry Multi-parameter flow cytometry was performed to measure percentages of different B cell, T cell, NK cell, and NKT cell populations within the total lymphocyte population and subsets within populations. Blood was drawn from the inner cubital vein and collected in 10 mL lithium heparin Vacutainer tubes (BD, Plymouth, UK). All subsequent steps were performed at 4˚C. Whole blood (1.5 mL) was centrifuged at 2000 g for 15 min, and plasma supernatant was aliquotted and stored at -20°C for anti- hepatitis B antibody titer analysis. The remaining whole blood was separated into two 50 mL tubes and erythrocytes were lyzed by incubating twice with 40 mL of ammonium chloride per tube for 10 min. Cell pellet

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CHAPTER 6. LONG CHAIN INULIN-TYPE FRUCTANS BUT NOT SHORT CHAIN INULIN-TYPE FRUCTANS ENHANCE HEPATITIS B VACCINATION RESPONSE IN YOUNG ADULTS. was collected by centrifuging for 5 min at 1800 g. After washing the cell pellet twice with 15 mL FACS buffer (2% fetal bovine serum in phosphate buffered saline, FBS in PBS), cells were counted on a coulter counter (Beckton Dickinson, the Netherlands) and 1 x 106 cells per well were transferred to a round bottom 96 wells plate. After pelleting the cells for 5 min at 1800 g and discarding the supernatant, cell pellets were resuspended in 50 µl of blocking buffer (20% normal rat serum, Jackson laboratories, in FACS buffer) and incubated for 20 min. Cells were pelleted as described above, resuspended in 50 µl of extracellular antibody mix consisting of extracellular antibodies, 5% normal rat serum, and FACS buffer (antibodies are listed in Table 1-3), and were incubated in the dark for 30 min. After washing the cells which were stained for B cell and NK cell markers, twice with 200 µl FACS buffer per well, cells were incubated with 200 µL of FACS-lysing buffer (Beckton Dickinson BV, Breda, the Netherlands) for 30 min. Cells were then washed three times with FACS buffer, resuspended in 200 µl FACS buffer per well, and stored at 4°C in the dark until analysis. After incubation with the extracellular antibody mix, the cells stained for T cell markers were washed three times with 200 µl of permeabilization buffer (eBioscience, Vienna, Austria) per well. Cells were resuspended in 50 µl of intracellular blocking buffer (20% normal rat serum in permeabilization buffer) and incubated for 20 min. After pelleting the cells as described above and discarding the supernatant, cells were incubated with 50 µL of intracellular antibody mix, consisting of intracellular antibodies, 5% normal rat serum, and permeabilization buffer (antibodies are listed in Table 2), and incubated for 30 min. Cells were washed three times with permeabilization buffer, resuspended in 200 µl of FACS buffer, and stored at 4°C in the dark until analysis on an LSR II flow cytometer (Beckton Dickinson BV). The corresponding isotype control antibodies were purchased from the same company as the target antibodies, and isotype stainings were used to set positive gates, using 1 % margins. UltraComp eBeads (eBioscience, Vienna, Austria) were applied to set the appropriate compensation values for each antibody panel. FlowJo VX software (FlowJo, Oregon, USA) was used to analyze lymphocyte subsets. Basal sampling at day 0 for every individual allowed repeated measures analysis on the flow cytometry data. Flow cytometry gating strategies are described in Figure 3.1, 3.2 and 3.3. Within the B cell population we used the markers CD19, CD21, CD27, CD38, IgA, IgD, IgG, and IgM to identify naïve non-class switched B cells, class switched memory B cells (IgG+ and IgA+), non-class switched memory B cells, transitional B cells, and plasmablasts/plasma cells. Using

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CD3, CD56, and CD16 as markers we identified NK-, and NKT cells, and within the NK cell population we identified cytotoxic NK cells (CD56+,CD16hi)26 and cytokine producing NK cells (CD56hi,CD16dim or CD56hi,CD16-)26. We determined the percentage of CD161+ and CD335+ cells within the cytotoxic-, and cytokine producing NK cells. CD161 is a cell surface marker which can be present on different subsets of NK cells27. Ligation of CD161 is known to cause activation of Pi3K, PkB, Akt and ERK pathways and is important in the regulation of NK and NKT cell function27. CD335 is a natural cytotoxicity-triggering receptor also known as PCR1 or NKp46 28. We used CD3 and CD8 as markers to distinguish Th cells and CTLs, and within the Th cell population we identified memory cells (CD45RO+), Th1 cells (TBET+), Th2 cells (CD294+), Th17 cells (RoRγT+), and FoxP3+ Th cells which may represent regulatory cells. It is generally believed that for the time frame following challenge with a virus or a viral vaccine, human CD8+ memory cells are principally found within the CD45ROhi population 29, therefore we also gated and analyzed this population.

6.5 Anti-HBsAg titer analysis Anti-HBsAg titers were analyzed at day 0, 14, 21, and 35. Per sample, 500 μl of plasma was aliquotted in Architect tubes (Abbott, Illinois, U.S.A.), and analyzed for Anti-HBsAg titers using an Architect Immunoassay Analyzer (Abbott Diagnostics) following the manufacturer’s instructions.

Table 1. Antibodies applied for flow cytometry of B lymphocytes. Antibody and label company cat# dilution CD19-PE ITK, Biolegend 302208 10x CD21-PE-Cy7 ITK, Biolegend 354912 100x CD27-BV421 ITK, Biolegend 302824 50x CD38-A700 ITK, Biolegend 303524 100x IgA-FITC DAKO F0188 20x IgD-APC ITK, Biolegend 348222 20x IgG- PerCP-Cy5.5 ITK, Biolegend 409312 7.5x IgM-BV605 ITK, Biolegend 314524 10x

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Figure 3.1. Gating strategies for peripheral blood B lymphocyte populations. Single cells are gated using FSC-H vs. FSC-A scatter plot (A). Single cells are gated to a FSC vs. SSC plot, and lymphocyte gate is set (B). Within the lymphocyte population, B cells (CD19+ ) are identified as a separate population (C). The CD21 isotype is used to set the gate margin on 1% positive cells (D) and this gate is copied to the CD21 stained sample (E, upper plus lower gate), identifying CD21+ cells. Within this population, the CD27 isotype is used to set the gate margin for 1% positive cells (F, upper gate) and the CD27- population (F, lower gate) and this gate is copied to the CD27 stained sample (G). Within the CD27- population (G), IgD isotype is used to set the gate margin on 1% positive cells (H) and this gate is copied to the IgD stained sample (I), identifying the naïve non-class switched B cells. Within the CD19+ population, CD27 isotype is used to set the gate margin on 1% positive cells (J) and this gate is copied to the CD27 stained sample (K), identifying CD27+ cells. Using the IgD isotype, the 1% positive gate and negative gate are set (L), and the negative gate is copied to the IgD stained sample (M) to identify the IgD- class switched memory population. Within this

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CHAPTER 6. LONG CHAIN INULIN-TYPE FRUCTANS BUT NOT SHORT CHAIN INULIN-TYPE FRUCTANS ENHANCE HEPATITIS B VACCINATION RESPONSE IN YOUNG ADULTS. population, isotypes are used to set 1% positive gates for IgG (N) and IgA (Q), which are copied to IgG and IgA stained samples to identify IgG+ and IgA+ class switched memory cells (panel O and R). The isotype gate set in panel L is copied to the IgD stained sample to identify CD19+ CD27- IgD+ cells (P). Within this population, the IgM isotype is used to set a 1% positive gate (S), which is copied to the IgM stained sample to identify the IgM+ non- class switched memory B cells (T). Within the CD19+ population, the CD38 isotype is used to set a 1% positive gate (U), which is copied to the CD38 stained sample to identify CD38+ and CD38hi cells (V). Within the CD38hi population the IgM isotype is used to set a 1% positive gate (W), which is copied to the IgM stained sample to identify IgMint cells (X*) and the IgMhi transitional B cell population (X**). The CD21lo population as set in panel E is copied to this IgMint cell population in panel X, identifying the plasmablasts and plasma cells (Y).

Figure 3.2. Gating strategies for peripheral blood NK and NKT lymphocyte populations. Single cells and lymphocytes are gated as described for B lymphocytes (A,B). Within the lymphocyte population, CD56+ CD3+ cells are gated (C, **), indicating the NKT cells. Within the lymphocyte population, the CD3- population is gated (C,*), and plotted in a panel with CD56 on the x-axis and CD16 on the y-axis (D). In panel D, left, the cytokine producing cells are gated [CD56hi CD16+], and the cytotoxic cells are also gated, panel D, right [CD56int CD16hi]. Within these populations, isotype for CD161 (E and I), and isotype for CD335 (G and K) were used to set 1% positive gates, which were copied to the CD161 stained samples (F and J) and to the CD335 stained samples (H and L) respectively.

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Figure 3.3. Gating strategies for peripheral blood T lymphocyte populations. Single cells and lymphocytes are gated as described for B lymphocytes (A, B). The T cell population within lymphocytes are gated in a CD3 vs. CD8 plot (C). CTLs are identified and gated based on expression of CD3 and CD8 (D, upper population). The CD45RO isotype is used to set the gate margin on 1% positive CTLs (E). This gate is copied to the CD45RO stained sample, now demonstrating the CD45RO+ population, and the upper cell cluster now indicates CD45ROhi cells (F). Th cells are identified based on expression of CD3 and no expression of CD8 (D, lower population). Isotype controls are used to set the gate margins on 1% positive cells for CD45RO, TBET, and RoRγT (G, I, L). These gates are copied to the CD45RO-, TBET-, and RoRγT stained samples, now demonstrating the positive populations (H, J ,M). A cross-gate is applied to identify FoxP3+ Th cells (*) and CD294+ Th2 cells (**) in panel K.

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Table 2. Antibodies applied for flow cytometry of NK/NKT lymphocytes. Antibody and label company cat# dilution CD3-PerCP ITK, Biolegend 300428 30x CD16-E450 eBioscience 48-0168-42 10x CD56-APC eBioscience 17-0569-42 25x CD335-PE ITK, Biolegend 331908 30x CD161-PE-Cy7 eBioscience 25-1619-42 20x

Table 3. Antibodies applied for flow cytometry of T lymphocytes. Antibody and label company cat# dilution CD3-Pacific Blue BD 558117 25x CD8-PerCP BD 345774 25x CD45RO-biotin Biolegend 304220 25x Streptavidin- Pacific Orange LifeTechnologies S32365 100x FoxP3-APC eBioscience 17-4776-42 25x TBET-PE-Cy7 eBioscience 25-5825-80 150x CD294-PE Miltenyi 130-098-879 40x RoRγT-PE eBioscience 12-6981-80 50x

Statistical analysis GraphPad Prism 5.0 was used for statistical analysis of all data. Flow cytometry data sets were analyzed for normal distribution using a d’Agostino Pearson test. To test for anti-HBsAg antibody titer development per supplement, statistical significance levels were determined with a Friedman test and Dunn’s multiple comparison test, comparing T14, T21, and T35 to T0 (basal samples), and Mann Whitney test was used for differences between supplements. Repeated measures ANOVA and Tukey’s multiple comparison test or a Friedman test and Dunn’s multiple comparison test were used to analyse the flow cytometry data, comparing T14, T21, and T35 to T0 (basal samples). P-values < 0.05 were considered statistically significant, and p = 0.1 was considered representative for a statistical trend.

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Results

6.6 Supplementation with DP10-60 fructans increased anti-HBsAg antibody production compared to supplementation with DP2-25 fructans and placebo Effects of nutritional supplementation with DP10-60 fructans vs. DP2-25 fructans on vaccination efficacy were studied by analyzing vaccine-induced anti-HBsAg antibodies in peripheral blood plasma samples of supplemented subjects using Architect Immunoassay analysis (Figure 4). Subjects supplemented with DP10-60 fructans developed antibody titers from time point T21 (0.6 ± 0.34 IQR) and reached significantly elevated levels at time point T35 compared to basal samples at T0 (2.93 ± 0.75 IQR, p < 0.05). In contrast, in plasma of subjects supplemented with DP2-25 fructans or placebo, no increases in antibody titers were observed at T35 as compared to basal samples at T0. Individuals developing an antibody titer against any vaccination, which is above a predetermined threshold value are indicated as so-called responders, vs. individuals who do not reach titers above this value, which are indicated as non-responders. This threshold value for the anti-HBsAg applied in the present study is generally set at 10 IU/mL 25. There we no responders in the placebo groups and the DP2-25 fructan group at T35. This was different in the DP10-60 fructan group in which two responders were present at time point T35. These results combined, demonstrate that supplementing young adults with a daily single bolus of 8 g DP10-60 fructans in the 14-day period around the first injection enhanced the efficacy of the anti- hepatitis B vaccination.

6.7 Flow cytometry analysis of peripheral blood B cell subsets Peripheral blood lymphocyte subsets of the supplemented individuals were analyzed by multi-parameter flow cytometry to study the effects of different DP fructans. Specifically, we were interested whether changes in T cell, B cell, NK cell, and NKT cell subsets were induced. As B cells are essential in mounting antibody responses against vaccines, we will first describe the results of B cell subsets in peripheral blood of subjects in the experimental groups. The subsets and their corresponding markers of identification are summarized in Table 1, gating strategies are shown in Figure 3, and results of B lymphocyte flow cytometry are shown in Figure 5. The percentage of B cells within the lymphocyte population was

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50 50 50 A B C 30 ** 30 30

10 10 10 10 10 10 8 8 8 6 6 6 4 4 4

anti-HBsAG titer anti-HBsAG 2 2 2 0 0 0 T0 T14 T21 T35 T0 T14 T21 T35 T0 T14 T21 T35 DP10-60 DP2-25 PLACEBO

7 D p 0.1 6 * DP10-60 5 DP2-25 PLACEBO 4

anti-HBsAG titer (IU/mL) titer anti-HBsAG 0 T0 T14 T21 T35

Figure 4. Anti-HBsAg antibody titer development in inulin-type fructan and placebo supplemented individuals in time. Statistical significance levels were determined with a Friedman test and Dunn’s post test for individual supplements to test for titer development compared to basal samples at T0 (Figure A-C), and Mann Whitney for differences between supplements (Figure D). Median and IQR of the anti-HBsAg titers are plotted in IU/mL. Supplements were dosed at 8g/d for 14 consecutive days around vaccination (n=13, n=13, and n=14 respectively for the supplemental groups DP10-60, DP2-25, and placebo). P = 0.1 indicates a statistical trend, * represents statistical difference with p < 0.05, and ** represents statistical difference with p < 0.01. analysed, and subsequently, the percentage of the following populations within the lymphocyte population or within the B cell population were analysed and compared to basal samples at T0 ; 1) Naïve non-class switched B cells [CD19+ CD21+, CD27- IgD+], 2) Class-switched memory B cells [CD19+ CD27+ IgD-; within this population the percentages of IgA+

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Figure 5. B cells and B cell subsets as percentages of lymphocytes, or within subsets in time, plotted per supplement. A) DP10-60 fructans, B) DP2-25 fructans, C) placebo. A Friedman test and a Dunn’s post test were used to analyze time effect per supplement. Median and IQR are plotted as percentage of the indicated cell populations, n=13, 13, and 14 for supplement DP10-60, DP2-25, and placebo respectively. * represents statistical difference with p < 0.05, and ** represents statistical difference with p < 0.01.

and IgG+ cells were analysed], 3) Non-class switched memory B cells [CD19+ CD27+ IgD+ IgM+], 4) transitional B cells [CD19+ CD38hi IgMhi], and 5) plasmablasts or plasma cells [CD19+ CD38hi IgMint CD21lo].

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The percentages of B cells in the total lymphocyte population did not change as compared to basal samples (Figure 5.I). Naïve non-class switched B cells (Figure 5.II), class switched memory B cell populations (Figure 5.III), and non-class switched memory B cells (data not shown) did not differ in time compared to basal samples as percentage of lymphocytes or B cells. The percentage of IgM+ cells in the non-class switched memory B cell population (Figure 5.IV) was increased for the placebo group at T21 compared to the basal samples (p < 0.05), but in the DP10-60 group and DP2-25 group no changes were observed as compared to basal samples. The percentage of transitional B cells and the percentages of plasmablasts or plasma cells did not differ in time as percentage of lymphocytes or B cells (Figure 5.V and 5.VII). However, in the DP10-60 fructan group and the placebo group, the percentages of IgMhi cells within the CD38hi transitional B cells were significantly increased at T14 and T21 as compared to basal samples (p < 0.05). This effect was absent in the DP2-25 fructan group (Figure 5.VI).

6.8 Flow cytometry analysis of peripheral blood NK-, and NKT cell subsets Human NK cells (CD56+ CD3-) are part of the first line of defense against viral pathogens and their activation can modulate the outcome of the adaptive immune response 30. Similar to NK cells, NKT cells (CD56+ CD3+) are also implicated in the response against hepatitis B vaccine antigens 30. The subsets and their corresponding markers of identification are summarized in Table 2, gating strategies are depicted in Figure 3, and results of NK lymphocyte flow cytometry are depicted in Figure 6. Within the lymphocyte population and within the NK cell population, the percentages of cytokine producing (CD56hi CD16int) or cytotoxic (CD56int CD16hi) cells were determined (Figure 6.I and 6.IV), and within the cytotoxic and cytokine producing cells, the percentages of CD161+ and CD335+ cells were analysed. No differences were observed for either cytotoxic or cytokine producing NK cell populations as percentage of the lymphocyte population. In the DP2-25 group and placebo group (Figure 6.II), an increased effect was observed for CD161+ cytokine producing NK cells at all time points compared to basal samples (T0) but in the DP10-60 group this effect was only observed on time points T21 and T35 compared to basal samples (p < 0.05). No changes were observed for CD161+ cytotoxic NK cells as compared to basal samples irrespective of the treatment. The percentage of CD335+ cells did not differ in time within

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CHAPTER 6. LONG CHAIN INULIN-TYPE FRUCTANS BUT NOT SHORT CHAIN INULIN-TYPE FRUCTANS ENHANCE HEPATITIS B VACCINATION RESPONSE IN YOUNG ADULTS. either NK cell population. In the DP10-60 group (Figure 6.VII), NKT cells tended to show a decrease on time point T14 compared to basal samples, expressed as percentage of the lymphocyte population (p = 0.1), but no changes were observed in the DP2-25 and placebo group.

Figure 6. Percentages of cytokine producing NK cells and cytotoxic NK cells, and percentages of CD161+ and CD335+ cells within these populations, and NKT cells within the lymphocyte population in time, plotted per supplement. A) DP10-60 fructans, B) DP2-25 fructans C) placebo. Repeated measures ANOVA and Tukey’s post test or a Friedman test and a Dunn’s post test were used to analyze time effect per supplement. Median and IQR are plotted as percentage of the indicated cell populations, n=13, 13, and 14 for supplement DP10-60, DP2-25, and placebo respectively. P = 0.1 indicates a statistical trend, * represents statistical difference with p < 0.05, ** represents statistical difference with p < 0.01, and *** represents statistical difference with p < 0.001.

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6.9 Flow cytometry analysis of peripheral blood T cell subsets Gating strategies are depicted in Figure 3, and T cell antibodies and labels are listed in Table 4. Results of T lymphocyte flow cytometry are shown in Figure 7. Within the T cell population, we analysed the percentages of Th1 cells, Th2 cells, FoxP3+ Th cells, Th17 cells, Th memory cells, and CTL memory cells. Percentages of Th2 cells, FoxP3+ Th cells, and Th17 cells at T35 did not differ from basal samples at T0 (Figures 5.II, 5.III, and 5.IV). At time point T35, the percentage of TBET+ (Th1) cells in the DP10-60 fructan group was significantly increased as compared to basal levels (p < 0.05), but this effect was not observed in the DP2-25 group or placebo group (Figure 5.I). Compared to basal samples at T0, the percentage of CD45ROhi CTLs expressed as percentage of CTL memory cells was increased at time point T14 and T35 in the DP10-60 group (p < 0.05), and at time point T35 in the DP2-25 group (p < 0.05), but no significant increases were observed in the placebo group (Figure 5.V). In addition to the CTLs, Th cells also typically demonstrated changes in the CD45ROhi population. However, only the placebo group demonstrated significant increases in CD45ROhi Th cells as percentages of the Th memory population (Figure 5.VI), at time point T14 and T35 as compared to basal samples at T0 (p < 0.05).

DIscussion

Previous studies from others and us have shown that inulin-type fructans can impact immunity by either serving as microbiota accessible fiber 17 or by directly binding to immune cells 20, but scientific evidence in humans of related immunological benefits were largely lacking. Building on in vitro results in our studies with inulin-type fructans, we hypothesized that DP10-60 fructans would stimulate vaccine responses in vivo due to their predominant induction of proinflammatory cytokines in PBMCs, including a relatively low IL-10/IL-12 ratio20, in combination with their strong ability to activate TLR220 31. We observed that supplementation with DP10-60 fructans significantly enhanced the titer response (T35 of the study) as compared to the DP2-25 fructan supplemented group, and that a strong increased trend was present as compared to the titer development in the placebo group. Another observation that supports the immune stimulating effects was the identification of two responders in the DP10-60 group vs. no responders in either the DP2-25 group or the placebo group. Inulin- type fructans have been studied in several infant vaccination trials, but in

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CHAPTER 6. LONG CHAIN INULIN-TYPE FRUCTANS BUT NOT SHORT CHAIN INULIN-TYPE FRUCTANS ENHANCE HEPATITIS B VACCINATION RESPONSE IN YOUNG ADULTS. the majority the fructans were only studied in combination with GOS and/or pectic oligosaccharides. In one study though, long term supplementation with a mixture of oligofructose/inulin, i.e. short chain inulin-type fructans combined with long chain inulin-type fructans,

Figure 7. T cell subsets expressed as percentages of Th cells or T memory cells in time, plotted per supplement. A) DP10-60 fructans, B) DP2-25 fructans C) placebo. A Friedman test and a Dunn’s post test were used to analyze time effect per supplement. Median and IQR are plotted as percentage of the indicated cell populations, n=13, 13, and 14 for supplement DP10-60, DP2-25, and placebo respectively. * represents statistical difference with p < 0.05.

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CHAPTER 6. LONG CHAIN INULIN-TYPE FRUCTANS BUT NOT SHORT CHAIN INULIN-TYPE FRUCTANS ENHANCE HEPATITIS B VACCINATION RESPONSE IN YOUNG ADULTS. enhanced the vaccination responses. In this study Saavedra et al. 22 observed an increase in blood IgG levels after measles vaccination in a 10 week supplementation study with oligofructose (OF)/inulin (7/3, 0,2 g/kg BW/d) in 7-9 months old infants. In a study by Duggan et al. 32 in which 6- 12 month old infants were supplemented with OF, i.e. only short chain inulin-type fructans (0.7 g/d), no effect was observed on antibody response after vaccination with H. influenza type B vaccine. Strikingly, other studies in infants with prebiotic mixtures did not induce vaccine potentiating effects 23 33. It should be noted that the applied fructans in these mixtures are often - if not always - of a short chain nature. Although this body of evidence is still relatively small, it is tempting to speculate that the DP10-60 fructans are indeed more suitable to apply for the purpose of potentiating vaccination programs. Dietary fibers can differ substantially in their molecular composition, and even within categories such as inulin-type fructans, different effects on the body and microbiota can be elicited15-19. To study the effect of different DP of fructans on changes in peripheral lymphocytes, we analyzed B cell-, T cell-, NK cell, and NKT cell subsets of supplemented individuals for their percentage of the total lymphocyte population, percentage of relevant subpopulations, and for differences in activation marker expression. Class-switching is one of the hallmarks of activation and maturation of B cells. If the peripheral blood is representative for the immune responses occurring after the vaccination, the increased titer response at T35 would be expected to coincide with a decrease in naïve B cells, and increases in transitional B cells or even plasma cells36. The percentage of transitional B cells which are in the process of maturation (CD38hi IgMhi) was significantly increased in the DP10-60 group and the placebo group for time points T14 and T21 compared to the basal samples but not in the DP2-25 group. Increased percentages of this population indicate that B cells are activated and stimulated to differentiate into antibody producing plasma cells, which is a functional objective of vaccination. The fact that this population was stimulated by DP10-60 fructan supplementation and not by DP2-25 fructan supplementation underscores the effective differences of these two supplements. The induction of memory B cells is important for the ability to mount an efficient secondary immune response and protection against infection upon encountering the relevant antigen36. In the non- class switched memory cells, the percentage of IgM+ cells was slightly increased for the placebo group at T21 compared to basal samples while the fructan groups did not. It is possible that the chosen time points for

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CHAPTER 6. LONG CHAIN INULIN-TYPE FRUCTANS BUT NOT SHORT CHAIN INULIN-TYPE FRUCTANS ENHANCE HEPATITIS B VACCINATION RESPONSE IN YOUNG ADULTS. sampling are too early after vaccination to observe the induction of B memory cells36 and that these cells arise after the 35 day period. NK and NKT cells did not demonstrate clear supplement dependent effects, suggesting that B cells and especially T cells may be more involved in the boosting of the vaccination response via dietary inulin supplementation, or that B cell and T cell effects may be better detectable in peripheral blood. Supplementation with DP10-60 fructans induced striking differences in T cell populations in time, the increased titer response for the long chain group on T35 was associated with an increased percentage of (TBET+) Th1 cells compared to basal samples. Contrary to Th1 cells, (CD294+) Th2 cells did not change in time for either treatment. These results suggest that in the DP10-60 group, a shifted Th1/Th2 balance may have been induced which was skewed towards Th1 cell responses. These results are corroborated by literature reports mentioned in several reviews 15-19, which subscribe that inulin-type fructans can shift the balance towards Th1 reactions instead of Th2 20 mediated responses. Although the in vitro results from our group , and others 34 led to expect increased Treg percentages at T35 in the DP2-25 group as compared to the DP10-60 group and the placebo group, no changes in regulatory T-cell populations were observed. Future vaccination studies may also include regulatory B cells in flow cytometry analysis, as these cells may also play a role in the success or absence of anti-HBsAg vaccination responses 35. It would be interesting to study whether these Bregs are stimulated by short chain inulin-type fructans as opposed to the Tregs which did not show increases in this study. Th17 populations also showed no changes in time or differences due to treatment. The induction of memory cells is important in the efficacy of vaccinations, as these cells are responsible for fast recognition of a second encounter with the relevant antigen and mounting an adequate immune response 36. The results indicate that both fructan treatments stimulate the induction of memory CD45ROhi CTLs but the placebo did not. On the other hand, the CD45ROhi Th cells were only enhanced in the placebo group at T35. It would be interesting in follow up studies to also include CD45RA and CCR7, to study the transitions into T cell memory or effector cells in more depth 37. The fact that we observed clear differences for the different DP compounds suggests that DP is an important factor which determines the reaction of immune cells in the body. This could be explained by several mechanisms. Upon consumption, inulin-type fructans of different DP may selectively stimulate different populations of the microbiota 38-40, and

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CHAPTER 6. LONG CHAIN INULIN-TYPE FRUCTANS BUT NOT SHORT CHAIN INULIN-TYPE FRUCTANS ENHANCE HEPATITIS B VACCINATION RESPONSE IN YOUNG ADULTS. these different populations could influence the immune system either in a stimulating or an attenuating manner 41. In addition, short chain fibers could be fermented into different products than long chain fibers, thus inducing different SCFA profiles in the intestine, qualitatively and quantitatively 17. However, most effects of SCFA on the immune system are attenuating 42, contrary to the immune stimulation results observed in the current study. It is more likely that the effects are the result of the sum of indirect and direct effects on immune cells in the intestine 17 19 20. It is a striking that an oral supplement can stimulate a systemic response to an intramuscular vaccination, and this warrants further studies into the way this process of antigen uptake and presentation can be impacted by orally taken supplements. Generally, the antigens of an intramuscular vaccination such as the applied hepatitis B vaccination, are thought to be detected by circulating dendritic cells, which then recruit other immune cells and migrate towards a draining lymph node, where antigen is presented to B-, and T cells followed by a primary immune response 36. Because of the natural surveillance function exerted by DCs, they circulate through the body and mount immune responses against antigens which are encountered. Ligation of innate immune receptors on DCs followed by antigen presentation toward effector cells, such as B cells, T cells, and NK cells locally, or in specialized lymphoid structures could be a mechanism which explains the induction of pro- and anti-inflammatory cytokines as observed in many prebiotic studies. Due to the fact that DCs can ‘sample’ the gut lumen 43 and they are migratory cells 44, they are one of the candidate cell types to mediate dietary fiber-induced immune effects occurring in the periphery. In conclusion, this is the first in vivo study which demonstrates a structure-function relation of a dietary fiber on a human immune response. We demonstrate that events in the gut by supplementing with a dietary fiber, have consequences for systemically induced changes in the immune system. The in vivo immunostimulatory potential of long-chain enriched inulin-type fructans subscribes an important nutritional health claim that these fibers can be beneficial for the immune system. Finally we feel that the use of a low efficacy vaccination model such as Hepatitis B may be more instrumental to demonstrate immunological effects of a nutritional supplement than the very efficient vaccination models 22 23 32 33. These human studies can be done with relatively low numbers of subjects and still with a high statistical power.

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Acknowledgements

The research was jointly financed by the European Union, European Regional Development Fund and the Ministry of Economic Affairs, Peaks in the Delta, the Municipality of Groningen, the Provinces of Groningen, Fryslan and Drenthe, as well as the Dutch Carbohydrate Competence Center (CCC WP2a), and by Royal Cosun.

Within the framework of the Carbohydrate Competence Center (CCC WP2a) to which Royal Cosun contributed both financially and intellectually, the data obtained in the course of this study and reported in this chapter/article are considered proprietary data of the parties to CCC WP2a, patent pending.

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xue ban = Journal of Central South University.Medical sciences 2011 Nov;36(11):1046-1051. (35) Garner-Spitzer E, Wagner A, Paulke-Korinek M, et al. Tick-borne encephalitis (TBE) and hepatitis B nonresponders feature different immunologic mechanisms in response to TBE and influenza vaccination with involvement of regulatory T and B cells and IL-10. Journal of immunology (Baltimore, Md.: 1950) 2013 Sep 1;191(5):2426-2436. (36) Siegrist CA. Vaccine Immunology. In: Plotkin S, Orenstein W, Offit P, editors. Vaccinese-book.: Elsevier;2013;17-18-36. (37) Carrasco J, Godelaine D, Van Pel A, et al. CD45RA on human CD8 T cells is sensitive to the time elapsed since the last antigenic stimulation. Blood 2006 Nov 1;108(9):2897- 2905. (38) Rumessen JJ, Bode S, Hamberg O, et al. Fructans of Jerusalem artichokes: intestinal transport, absorption, fermentation, and influence on blood glucose, insulin, and C- peptide responses in healthy subjects. The American Journal of Clinical Nutrition 1990 Oct;52(4):675-681. (39) Alles MS, Hautvast JG, Nagengast FM, et al. Fate of fructo-oligosaccharides in the human intestine. The British journal of nutrition 1996 Aug;76(2):211-221. (40) van de Wiele T, Boon N, Possemiers S, et al. Inulin-type fructans of longer degree of polymerization exert more pronounced in vitro prebiotic effects. Journal of applied microbiology 2007 Feb;102(2):452-460. (41) Smelt MJ, de Haan BJ, Bron PA, et al. Probiotics can generate FoxP3 T-cell responses in the small intestine and simultaneously inducing CD4 and CD8 T cell activation in the large intestine. PloS one 2013 Jul 4;8(7):e68952. (42) Meijer K, de Vos P, Priebe MG. Butyrate and other short-chain fatty acids as modulators of immunity: what relevance for health? Current opinion in clinical nutrition and metabolic care 2010 Nov;13(6):715-721. (43) Rescigno M, Urbano M, Valzasina B, et al. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nature immunology 2001 Apr;2(4):361-367. (44) Rivollier A, He J, Kole A, et al. Inflammation switches the differentiation program of Ly6Chi monocytes from antiinflammatory macrophages to inflammatory dendritic cells in the colon. The Journal of experimental medicine 2012 Jan 16;209(1):139-155.

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Chapter 7

General discussion

And future perspective

Leonie M. Vogt1

1 Department of Pathology and Medical Biology, Division Medical Biology, Groningen University, University Medical Center Groningen

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In a society in which the average lifespan is ever increasing, the importance of reaching old age in good health is evident. In this concept, well being is a natural result of healthy ageing, but there is also a large socio-economic advantage to be gained by reducing the annual health care costs. Nutrition is a very important component which can contribute to a healthy ageing process. Current opinions voice the essential role of sufficient fiber content in the daily diet to protect against a range of diseases. Although a collective health improving or protecting effect of different types of fibers is described, the individual mechanisms by which these types of fibers exert their health effects remain to be elucidated and warrant mechanistical studies into the individual structure-response relationships of different fiber types. The effects on the immune system are suggested to play an important role in dietary fiber mediated health effects. As a vital component of the body, the immune system is responsible for homeostasis by defending the body against pathogens. In addition, it is charged with the duty of maintaining a peaceful co-existence with the commensal microorganisms located in our intestines. The intestines can be considered as the most important immunological organ of the body as it harbors around 70 percent of our total immune cell population. It is constantly active in analyzing the status of the gut lumen by screening for ‘good’ or ‘bad’ signals in the form of commensal microbiota species vs. pathogenic organisms and toxins. Besides through continuous action of specialized immune cells, a tightly interconnected monolayer of epithelial cells prevents the external environment of the lumen from protruding into the surrounding tissue of the lamina propria. Continuous leakage of luminal content into surrounding tissue and into the periphery due to a defective epithelial barrier is an unwanted phenomenon, called ‘leaky gut syndrome’. Several diseases have so far been linked to a leaky gut, and it is expected that more will follow, indicating the importance of maintaining the epithelial barrier function for disease prevention. Functional foods like prebiotic dietary fibers are aimed at stimulating a healthy gut microbiota composition by selectively stimulating lactobacilli and bifidobacteria, which coincides with stimulating host health. In addition to the well-known indirect health effects via these bacteria and their SCFA products, we propose that there is a third mechanism by which dietary fibers can cause immune modulation, i.e. by directly activating membrane receptors on intestinal immune cells and epithelial cells. In this thesis, the call for mechanistic studies on individual fiber types and their direct effects on the immune system and intestinal cell barrier function is addressed. In order to study

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CHAPTER 7. GENERAL DISCUSSION AND FUTURE PERSPETIVE. relevant parameters, first a technology platform needed to be established, which was done by using a well-studied type of dietary fiber, i.e. chicory root derived inulin-type fructans. After the extraction process of these fibers from chicory, the remaining root pulp is mainly used as a high fiber livestock feed. Due to the increased interest in dietary fibers, cellulose and pectins, two major fibers from this byproduct, were also studied using the established technology platform to evaluate their health effects for the potential use in functional food products.

7.1 Direct immunomodulatory effects of inulin-type fructans In Chapter 2 we demonstrate for the first time the proof of principle that inulin-type fructans directly activate immune cells. We found that signaling is dependent on the TLR adapter molecule MyD88, and therefore that this type of signaling is mediated via TLRs. In particular, TLR2 demonstrated dose-dependent activation upon cellular stimulation with inulin-type fructans, suggesting that this is the most important TLR by which inulin-type fructans induce immune signaling. Strikingly, the higher the average chain length of the formulation, the stronger the TLR2 response as measured by NF-κB activation. Although receptor studies specifically aimed at dietary fibers are still scarce, PRRs appear to be involved in these type of interactions, probably mimicking their responsiveness towards oligo-, or polysaccharides which occur in the form of pathogen associated molecular patterns. Examples of these interactions known so far are beta-glucans which are recognized by dectin-1 and complement receptor 3, and also cause immune modulation [1], beta- galactosides which are recognized by galectins [2], Mannose receptors on macrophages recognizing mannan oligosaccharides [3], and DC-sign which binds to Lewis Antigen glycans [4]. Broadening screening experiments by including these additional receptors and more types of dietary carbohydrate polymers and oligomers will provide additional information in future dietary fiber studies on their direct immunomodulatory properties. Blocking of TLR2 on human PBMCs was performed to study whether TLR2 was the most important receptor inducing the IL-10/ IL-12 cytokine responses in these cells (results not shown); however, the results showed much interindividual variation in the induced cytokine pattern, suggesting that the interactions of fructans with PBMCs could be more complex as compared to THP1 cells. PBMCs comprise several very different cell types, therefore a suggestion to get a better insight into the effects of fructans on individual cell types, may be to use cell sorting to

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CHAPTER 7. GENERAL DISCUSSION AND FUTURE PERSPETIVE. first separate the cell types and evaluate the induced immune effects per specialized cell type.

7.2 Direct immunomodulatory effects of other chicory root dietary fibers To address whether other dietary fibers are also capable of activating PRRs, we studied cellulose (Chapter 4) and pectins (Chapter 5), which are two major components of chicory root pulp. We found that both fiber types induced NF-κB activation in THP1 cells, but the activation by cellulose was partly mediated through TLR/MyD88-dependent and partly through TLR/MyD88-independent signaling pathways. Other PRRs expressed by the THP1 cells and also signaling to NF-κB are likely to cause the TLR-independent effects and require further mechanistic studies. Candidate PRRs to study would be in the categories of C-type lectin receptors, RIG-like receptors, galectins, and other innate immune receptors known to interact with carbohydrate polymers and oligomers. The activating capacity of cellulose in itself was striking however, as many researchers apply cellulose as a placebo under the assumption that it is an inert compound which passes through the gastrointestinal tract without affecting the intestines. In follow up of these unexpected results, we performed a microchip array and identified several genes in the NF-κB-, and TLR pathways which were modulated in human PBMCs upon stimulation with cellulose. Subsequent reporter experiments also confirmed TLR2-mediated NF-κB activation as well as a biphasic dose- related pattern of TLR4-mediated NF-κB activation. Compared to inulin- type fructans, the TLR activation by cellulose could be categorized as moderate, but these results show promise for further experiments and the set up of chicory derived cellulose supplementation studies. The activation by pectins did prove to be fully MyD88-, and TLR- dependent, as the pectins induced no activation of the applied MyD88 deficient THP-1 cell line. Contrary to cellulose, pectins have been studied extensively for their health promoting and immunomodulatory effects, but also for pectic compounds the structure-function relationships and actual target receptors were not fully elucidated so far. Depending on their structural properties, pectins were capable of mounting a TLR2 response similar to long chain inulin-type fructans, indicating a strong and specific interation with this receptor. Pectins induced moderate TLR4 activation, however this was much less pronounced as compared to the activation of TLR2 and may have been due to interference of residual endotoxin traces in the original compound. On the other hand, if endotoxin is not selectively digested by the applied enzymes then the

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CHAPTER 7. GENERAL DISCUSSION AND FUTURE PERSPETIVE. complete abrogation of activation of THP1 cells by the pectins strongly suggests that the TLR4 activation as observed in the HEK cell lines was in fact not endotoxin related. To make a general statement about TLR4 activation by pectins, a broader range of pectin types should be studied, and the endotoxin content of the applied fibers should be carefully measured and documented in future studies. Ongoing experiments comprise characterization of immune and barrier effects of the remaining dietary fibers of which chicory root is comprised, such as the hemicellulose xyloglucan and acetylated pectins, to reveal the full potential of individual chicory root pulp components, and also encompass the study of the chicory root pulp when it is applied as a whole in a nutritional supplement.

7.3 Structure-function relationships: Chain length We aimed to elucidate whether and which structural properties of chicory root dietary fibers can modulate cellular immune responses (Chapter 2). One of these structural properties is fiber chain length. Upon studying inulin-type fructans, a specific chain length-dependency of immune responses was demonstrated, as the short chain-enriched inulin-type fructans induced a high, anti-inflammatory IL-10/IL-12 ratio in human PBMCs and the longer the average chain length of the inulin-type fructan formulation, the lower this ratio. A possible explanation for the difference in IL-10/IL-12 ratios can be deduced from our TLR reporter assays. Chain length-induced differences in activation, as observed in the experiments with inulin-type fructans, might be due to mechanistic differences in receptor interactions at the cellular surface by clustering smaller or larger numbers of the relevant receptors on the membrane, thereby creating a molecular complex which enhances signal transduction or alters the downstream outcome. This clustering mechanism has been described for LPS, clustering substantial numbers of TLR4 [5] and may be a relevant mechanism for other TLRs as well. A second explanation for the observed differences may be the interacting capacity of inulin-type fructans with cell membrane lipids in the membrane which was studied by Vereyken et al. [6]. Strikingly, the dynamics of these interactions are also chain length- dependent [7-9], and it may therefore be a plausible mechanism to explain different signaling outcomes. Another explanation for differences in TLR2-mediated signaling can be that TLR2 is known to be a promiscuous receptor which can partner with a spectrum of different co-receptors [10]. As a consequence, the outcome of TLR2 activation can be highly dependent on the type of co-

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CHAPTER 7. GENERAL DISCUSSION AND FUTURE PERSPETIVE. receptor which is involved [10]. Two important co-receptors of TLR2 are TLR1 and TLR6, which are both present on the HEK reporter cells which were applied in our studies, as well as on human PBMCs. Activation of TLR2 which is dimerized to TLR1 is considered to induce mostly proinflammatory signals [11], whereas the activation of TLR2 which is dimerized with TLR6 is more likely to induce an anti-inflammatory response [12]. The applied reporter assays in our studies are based on PRR-mediated NF-κB activation because NF-κB is an essential transcription factor involved in TLR-mediated innate immune responses against pathogens [13,14]. As such, the activation of NF-κB is a reliable read out for TLR-mediated activation. Nevertheless, TLR2 is also known in some cases to not only activate NF-κB, but also other transcription factors, including cAMP response element (CREB), which is involved in IL-10 expression [15,16]. Our results tend to suggest that short chain inulin-type fructans induce NF-κB but also other, more anti-inflammatory transcription factors, and that long chain inulin-type fructans destinctly and very strongly activate NF-κB in particular. Future studies with immune cell transcription factor screens of in vitro stimulated cells could provide confirmation of this explanation. Although quite specific mechanistic results were already acquired, at the same time these results call for further biochemical characterization of the interactions and dynamics of inulin-type fructans with membrane receptors and lipids. Literature reports regarding chain length dependent effects on the immune system or barrier function induced by other dietary fibers are relatively scarce. Oligosaccharides, which can be characterized as relatively short chain dietary fibers are often studied for their prebiotic and allergy-preventing properties in infants. The general idea behind the application of oligosaccharides in infant nutrition is to mimic the oligosaccharides which are present in breat milk, as these are strongly involved in the stimulation of a healthy intestinal microbiota and development of the immue system in infants. Oligosaccharides can be derived from a range of different backbone structures. Galactooligosaccharides (GOS) for example are often supplemented in a combination with inulin-type fructans and with or without (acidic) pectin oligosaccharides (pAOS). The typical mixture often applied in baby formula is comprised of a 9:1 ratio of GOS [17], which are short chain fibers with a DP ranging from 3 to 10, and long chain inulin, which ranges up to DP60. Since the allergy preventive effects of these infant supplements are aimed at stimulating the maturation of the immune system through beneficial bacteria, and skewing it into a relatively more Thelper1 oriented direction

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CHAPTER 7. GENERAL DISCUSSION AND FUTURE PERSPETIVE. and away from Thelper2 reactions which are allergy related [18,19], it would be highly interesting to know whether besides through prebiotic action, the TLR2 stimulation of immune cells and epithelial cells of infants is also involved in this beneficial skewing effects of dietary fibers, and inulin-type fructans in specific.

7.4 Structure-function relationships: Methyl esterification In the study of pectins we applied commercially available lemon pectins to confirm the principle of immunomodulatory properties of pectin (Chapter 5). In addition, these pectins are well characterized for their structure which is a linear backbone with different percentages of attached methyl groups. Literature on pectin-induced health effects describes that the amount and type of molecular groups esterified to the pectin backbone are of particular importance in determining the outcome of pectin challenges [20]. The observations that low DM (DM30) pectins only activated TLR2 at moderate concentrations, and high DM (DM56) pectins only activated TLR2 at higher concentrations confirm that pectin-induced activation is very sensitive to different experimental circumstances as also demonstrated and discussed by Popov et al. [20]. In addition, both DM30 and DM56 pectins only induced moderate activation, whereas DM74 pectins induced a relatively strong, and dose dependent TLR2 activation over the whole concentration range tested. When a comparison is drawn with the inulin-type fructan results for the strength of TLR2 activation and the anti-, or proinflammatory effects on the immune system, literature on pectin studies corroborated our results such that high DM pectins are generally characterized as immunostimulatory and low DM pectins tend to have immunoregulatory or anti-inflammatory properties [21]. A single study of pectins including cytokine analysis in pectin-stimulated PBMCs demonstrated opposite effects [22], suggesting that for a solid consensus more in vivo and in vitro experiments may still be required, however all circumstances of pectin experiments such as applied dose, duration of stimulation etc. should be carefully controlled, documented, and reported, as pectin studies can be very susceptible to these factors in changing the outcomes [20]. Other structural features of pectins which have been described in literature as an important determinants of the induced effects are the degree of acetyl groups attached to the pectin backbone or “Degree of Acetylation (DA)”, the branching and type of branches attached to the backbone, and also the molecular charge and acidity of the pectins [20]. These characteristics are interesting to include in future structure-response studies of pectins. Finally, to fully understand

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CHAPTER 7. GENERAL DISCUSSION AND FUTURE PERSPETIVE. the benefits of actual chicory root pectins for health, a detailed pectin content-, and structure profile of chicory root pulp is required as reported in the studies of Daas et al. [23,24], and ultra-pure pectin isolates of chicory can then be evaluated for their immunomodulatory and barrier protective effects using the technology platform we have described.

7.5 Barrier protective effects and TLR2 ligation With the activation of TLR2 by dietary fibers in mind, and considering that TLR2 is an essential molecule for the regulation of a proper intestinal barrier function [25], we hypothesized that dietary fibers confer protection on intestinal epithelial cell barrier function via Toll-like receptor 2 (TLR2). We studied whether chain-length and methylation differences influence this process. Inulin-type fructans exerted time-dependent and chain length–dependent protective effects on the T84 intestinal epithelial cell barrier mediated via TLR2. These results suggest that TLR2 located on intestinal epithelial cells could be a target of dietary fiber–mediated health effects through modulation of the intestinal barrier function. For this particular type of fiber, the short chain formulations actually proved efficient in barrier protection whereas the long chain inulin-type fructans showed little or no protection against PMA. The effects on barrier function relating to different degree of methyl esterification (DM) of pectins were not as straightforward; 30DM and 74DM pectins induced a strong barrier protective effect, whereas 56DM pectins induced only moderate protection of T84 TEER. Cellulose was also included in these ECIS experiments, however it did not exert any effects on T84 barrier function. Results of these three fiber types raise the question why moderate activators of TLR2 such as short chain inulin-type fructans would protect the barrier function, but stronger TLR2 activators in the form of long chain inulin-type fructans did not show this effect. Moreover, as a moderate TLR2 activator, cellulose would be expected to induce protection, which was not observed. And finally, both low DM as well as high DM pectins protected the barrier function. For now, the only explanation for these apparently contradictive outcomes is that there are fundamental differences in the way these fibers interact with the cellular receptors and that they show very distinctive and compound-specific effects. Further mechanistic studies are required to explain these differences in barrier protective properties or lack thereof.

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7.6 Human studies: Innate and adaptive immunomodulatory effects of inulin-type fructans At the commencement of the first experiments documented in this thesis, The European Food Safety Authority (EFSA) described the status quo of inulin-type fructans as certainly being a prebiotic functional food, but that more studies were required to provide scientific evidence of related health benefits. Currently, anno 2015, the property of stimulating beneficial microbiota is supplemented with an accepted health claim stating that native chicory inulin beneficially effects blood glucose levels and bowel habit, but specific claims regarding stimulation of the immune system still require more scientific evidence and more studies are highly recommended. This evicence should comprise the stimulation of a functional parameter such as stimulation of vaccination efficacy. To address this call for in vivo evidence of immunomodulatory effects of inulin-type fructans we designed a human supplementation study with the aim to confirm these effects (Chapter 6). The observed IL-10/IL-12 ratios suggested that application of short chain inulin-type fructans would be most suitable in situations which call for anti-inflammatory regulation of immune responses, and that supplementation with long chain-enriched inulin-type fructans would be suitable for applications where the immune system needs to be boosted in its response to pathogens. We hypothesized that long chain enriched inulin-type fructan supplementation in the critical 14 day period around the first injection of an anti-Hepatitis B surface antigen vaccination would be able to boost the adaptive immune responses against this vaccine. Evidence confirming this hypothesis was best observed at the final sampling time point T35, which was 35 days after the start of fiber intake, 28 days after receiving the vaccination, and 21 days after the final supplement intake. Anti-HBsAg titers in plasma of supplemented subjects confirmed that in the long chain enriched inulin-type fructan group, the titer response was significantly enhanced as compared to short chain inulin-type fructans, and showed a clear increased trend (p=0.01) as compared to the placebo group. Moreover, the category of responders, i.e. subjects with an antibody titer above 10IU/mL counted two subjects in the long chain inulin-type fructan group vs. no responders in the placebo group or the short chain-enriched inulin-type fructan group. Inulin-type fructans have been studied in several infant vaccination trials, but in the majority the fructans were only studied in combination with GOS and/or pectic oligosaccharides. In one study though, long term supplementation with a mixture of oligofructose/inulin, i.e. short chain inulin-type fructans

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CHAPTER 7. GENERAL DISCUSSION AND FUTURE PERSPETIVE. combined with long chain inulin-type fructans, enhanced vaccination responses. In this study Saavedra et al. [26] observed an increase in blood IgG levels after measles vaccination in a 10 week supplementation study with OF/inulin (7/3, 0,2 g/kg BW/d) in 7-9 months old infants. In a study by Duggan et al. [27] in which 6-12 month old infants were supplemented with OF, i.e. only short chain inulin-type fructans (0.7g/d), no effect was observed on antibody response after vaccination with H. influenza type B vaccine. Strikingly, other studies in infants with prebiotic mixtures did not induce vaccine potentiating effects [28,29]. It should be noted that the applied fructans in these mixtures are often - if not always - of a short chain nature. Although this body of evidence is still relatively small, it is tempting to speculate that the long chain inulin-type fructans are indeed more suitable to apply for the purpose of potentiating vaccination programs. To relate our findings of increased antibody titers to changes in peripheral lymphocytes, we analyzed B cell-, T cell-, and NK cell-subsets of supplemented individuals for their percentage of the total lymphocyte population, percentage of the relevant subpopulations, and for differences in activation marker expression. Supplementation with long chain inulin-type fructans induced striking differences in T cell populations in time, the increased titer response for the long chain group on T35 was associated with an increased percentage of TBET+ Thelper1 cells compared to basal samples, and this percentage was also significantly increased compared to all time point measurements of the placebo group. Although increased in time, at T35, the percentage of CD294+ Thelper2 cells was smallest in the long chain enriched inulin-type fructan group, compared to the short chain enriched inulin-type fructan group, and placebo group. These results suggest that in the long chain group a shifted Th1/Th2 balance was induced which was skewed towards Th1 cell responses. These results are corroborated by literature reports mentioned in Chapter 1, which demonstrate that inulin-type fructans can exert stimulation of Thelper 1 reactions and prevent Thelper2 mediated allergic responses. The induction of memory cells is important in the efficacy of vaccinations, as these cells are responsible for fast recognition of a second encounter with the relevant antigen and mounting an adequate immune response [30]. The percentage of memory CD45ROhi Th cells was increased for the long chain inulin-type fructan group as well as for the placebo group at T35, but not for the short chain inulin-type fructan group. Although the in vitro results from our group described in Chapter 2, and

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CHAPTER 7. GENERAL DISCUSSION AND FUTURE PERSPETIVE. results from literature [31] led to expect increased Treg percentages at T35 in the short chain inulin-type fructan group as compared to the long chain inulin-type fructan group and the placebo group, no changes in regulatory T-cell populations were observed. Th17 populations also demonstrated no changes in time or differences due to treatment. Thus, long chain inulin-type fructans as well as the placebo stimulate Th memory cells, but this result could also be regarded from a different viewpoint, in the way that the induction of Memory cells was actually inhibited in the short chain group. The significant increase in Thelper1 cells appears to be the parameter which can be linked directly to the increased vaccination reponse as it was significantly increased compared to both the short chain group and the placebo group. Changes in B cell populations were significant, but not as straightforward as for the T cell populations. Class-switching is one of the markers indicating activation and maturation of B cells. Strikingly in the B cell compartment, the percentage of IgM+ cells indicating the non-class switched memory B cell population was decreased for the long chain group at T35 compared to basal samples. Upon class switching, B cells can become IgD- and IgA+ or IgG+. And although no significant changes were observed for the total class switched IgD- subpopulation in the long chain group and the placebo group, the relative contribution of IgG+ cells within the IgD- population in fact were increased for the long chain enriched inulin-type fructan group at time point T21 as compared to basal samples. Finally, the percentage of transitional B cells which are in the process of maturation (CD38hi IgMhi) was significantly increased in the long chain group and the placebo group for time points T14 and T21 compared to the basal samples but not in the short chain group. Increased percentages of this population indicates that B cells are activated and stimulated to differentiate into antibody producing plasma cells, which is one of functional objectives of vaccination. The fact that this population was stimulated by long chain supplementation and not by short chain supplementation is a strong evidence of the effective differences of these two supplements. NK and NKT cells did not demonstrate clear supplement dependent effects, indicating that B cells and especially T cells are more involved in the boosting of the vaccination response via dietary inulin supplementation. Future vaccination studies may also include regulatory B cells in flow cytometry analysis, as these cells may also play a role in the success or absence of anti-HBsAg vaccination reponses [32]. It would be interesting to study whether these Bregs are stimulated by short chain

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CHAPTER 7. GENERAL DISCUSSION AND FUTURE PERSPETIVE. inulin-type fructans as opposed to the Tregs which did not show increases in this study. We observed clear results from this human study, and long chain inulin-type fructans are now established to have in vivo immunostimulatory properties in humans. This pioneer study can provide important clues for further studies into the mechanisms how long chain inulin-type fructan supplementation leads to potentiation of the response against the hepatitis B vaccine. Generally, the antigens of an intramuscular vaccination such as the applied hepatitis B vaccination, are thought to be detected by circulating dendritic cells, which then recruit other immune cells and migrate towards a draining lymph node, where antigen is presented to B-, and T cells followed by a primary immune response [30]. The exact mechanism by which inulin-type fructan supplementation boosts this process remains to be studied, but can include effects on the gut microbiota and SCFA, as well as effects on dendritic cells circulating through the intestine and the periphery. Because of the natural surveillance function exerted by DCs, they circulate through the body and mount immune responses against antigens which are encountered. Ligation of innate immune receptors on DCs followed by antigen presentation toward effector cells, such as B cells, T cells, and NK cells locally, or in specialized lymfoid structures could be a mechanism which explains the induction of pro- and anti-inflammatory cytokines as observed in many prebiotic studies. Due to the fact that DCs can ‘sample’ the gut lumen and they are migratory cells, they are one of the candidate cell types to mediate dietary fiber-induced immune effects occurring in the periphery. The role of dendritic cells in these processes are still to be confirmed, which should start with in vitro culture of cell lines which are differentiated into cells with a dendritic phenotype and analysis of the effects of inulin-type fructans on the cytokine production, the antigen presentation potency of these cells towards effector cells and the skewing of effector cell responses. In addition, barrier measurements in vivo are neccesary to corroborate the statements made in Chapter 3; these experiments are relatively easy and non-invasive for test subjects, as they can be performed by means of lactulose-mannitol tests during dietary supplementation trials. These tests are highly recommended for future dietary fiber supplementation studies.

7.7 Future perspective The studies presented in this thesis demonstrate that chicory root dietary fibers exert direct effects on immune cells and intestinal epithelial cell

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CHAPTER 7. GENERAL DISCUSSION AND FUTURE PERSPETIVE. barrier function. We demonstrate that not only the primary product of chicory root, i.e. inulin-type fructans, posses valuable nutritional value but that this also holds true for fiber components of the remaining chicory root pulp after extraction of the inulin. Moreover, the effects of inulin- type fructans do not only comprise modulation of innate immune responses but can range as far as adaptive responses in the periphery. We describe a technology platform of in vitro studies and human studies which can be applied for future evaluation of beneficial effects of many more dietary fibers, or other nutritional compounds of interest.

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cumulative incidence of atopic dermatitis in infants at risk. J Pediatr Gastroenterol Nutr. 2006;42: E5. 20. Popov SV, Ovodov YS. Polypotency of the immunomodulatory effect of pectins. Biochemistry (Mosc). 2013;78: 823-835. 21. Popov S, Markov P, Popova G, Nikitina I, Efimova L, Ovodov YS. Anti-inflammatory activity of low and high methoxylated citrus pectins. Biomedicine and Preventive Nutrition. 2013;3: 59-60-63. 22. Salman H, Bergman M, Djaldetti M, Orlin J, Bessler H. Citrus pectin affects cytokine production by human peripheral blood mononuclear cells. Biomed Pharmacother. 2008;62: 579-582. 23. Daas PJH, Voragen AGJ, Schols HA. Characterization of non-esterified galacturonic acid sequences in pectin with endopolygalacturonase. Carbohydrate Research. 2000;326: 120-121-129. 24. Daas PJH, Boxma B, Hopman AMCP, Voragen AGJ, Schols HA. Nonesterified galacturonic acid sequence homology of pectins. Biopolymers. 2001;58: 1-2-8. 25. Cario E. Barrier-protective function of intestinal epithelial toll-like receptor 2. Mucosal Immunol. 2008;1 Suppl 1: S62-6. 26. Saavedra JM, Tschernia A. Human studies with probiotics and prebiotics: Clinical implications. Br J Nutr. 2002;87 Suppl 2: S241-6. 27. Duggan C, Penny ME, Hibberd P, Gil A, Huapaya A, Cooper A, et al. Oligofructose- supplemented infant cereal: 2 randomized, blinded, community-based trials in peruvian infants. Am J Clin Nutr. 2003;77: 937-942. 28. Stam J, van Stuijvenberg M, Garssen J, Knipping K, Sauer PJ. A mixture of three prebiotics does not affect vaccine specific antibody responses in healthy term infants in the first year of life. Vaccine. 2011;29: 7766-7772. 29. van den Berg JP, Westerbeek EA, van der Klis FR, Berbers GA, Lafeber HN, van Elburg RM. Neutral and acidic oligosaccharides supplementation does not increase the vaccine antibody response in preterm infants in a randomized clinical trial. PLoS One. 2013;8: e70904. 30. Siegrist CA. Vaccine immunology. In: Plotkin S, Orenstein W, Offit P, editors. Vaccines. e-book.: Elsevier; 2013. pp. 17-18-36. 31. Li J, Tan D, Liu H, Li K. CD4(+) CD25(+) FoxP3(+) T regulatory cells in subjects responsive or unresponsive to hepatitis B vaccination. Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2011;36: 1046-1051. 32. Garner-Spitzer E, Wagner A, Paulke-Korinek M, Kollaritsch H, Heinz FX, Redlberger-Fritz M, et al. Tick-borne encephalitis (TBE) and hepatitis B nonresponders feature different immunologic mechanisms in response to TBE and influenza vaccination with involvement of regulatory T and B cells and IL-10. J Immunol. 2013;191: 2426- 2436.

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Current consensus on a healthy diet is that sufficient daily dietary fiber intake can protect us from developing several diseases. The exact mechanisms by which this protection occurs are currently unknown but the effects on the immune system are suggested to play an important role. In addition to the indirect immune effects elicited by lactid acid bacteria and their SCFA products, we propose that there is a third mechanism by which dietary fibers can cause immune modulation, i.e. by directly activating membrane receptors on immune cells and epithelial cells. Although a collective health improving or protecting effect of different types of fibers is described, the individual mechanisms by which these types of fibers exert their health effects remain to be elucidated. The studies described in this thesis were aimed specifically to identify and characterize direct effects of different dietary fibers from chicory root. The acquired knowledge of chicory root dietary fibers can be applied in designing a healthy daily nutritional regimen and provides us with the tools to design chicory-derived functional foods for specific immune boosting purposes.

Chicory root has been known for its high content in one of the best characterized prebiotic fibers, inulin-type fructans. Chapter 1 provides a literature overview of the immune mediated health effects of inulin-type fructans. From studies that applied these fibers in colon cancer, chronic inflammatory diseases, vaccination efficacy, and prevention of infection and allergy, a large body of evidence with a range of immunomodulatory effects was gathered. These fructans are potent immunomodulating food components that hold many promises for prevention of disease. Recommended for future studies on immunological effects of inulin-type fructans and other dietary fibers is that the choice of immune markers is correlated with the particular condition that is being assessed, and the relevant clinical end-points should be clear. In case of patient studies, it should be mentioned whether immune markers are differentially expressed in disease and control populations. Due to the lack of experimental evidence, studies aimed at identifying direct effects of immune modulation through activation of gut DCs or other gut-residing immune cells are warranted. We hypothesize that by ligating receptors of the innate immune system such as TLRs, and NODs, dietary fiber supplementation can have substantial effects on the intestinal as well as the peripheral immune system. In addition, dietary fibers may also ligate PRRs expressed on gut epithelium, which could influence its barrier function.

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In Chapter 2 we describe that inulin-type fructans exert direct signaling upon contact with immune cells, and we discuss the role played by TLRs and NODs in this process. Human PBMCs were studied as model immune cells as they express many PRRs including TLRs and NODs. These PBMCs were stimulated with four inulin-type fructan formulations with different chain length profiles. Not only direct activation was observed by significant production of cytokines upon stimulation, but also that the produced cytokine patterns were dose-, and chain length dependent. Strikingly, short chain enriched inulin-type fructans induced a regulatory cytokine balance compared to long chain enriched inulin-type fructans as measured by higher vs. lower IL- 10/IL-12 ratios respectively. TLR reporter cell experiments demonstrated that fructan-induced signaling is highly dependent on TLRs and their adapter MyD88. From the follow up experiments in reporter cells overexpressing a single type of PRR per cell line, we concluded that TLR2 was prominently activated, while TLR4, 5, 7, 8, and NOD2 were mildly activated. These results indicate that inulin-type fructans indeed possess direct signaling capacity on human immune cells, and that this activation is dose-, chain length-, and predominantly TLR2 dependent.

TLR2 is an important receptor involved in intestinal barrier homeostasis. The observation that TLR2 is prominently involved in inulin- type fructan recognition prompted us to investigate whether these fibers improve or protect the epithelial barrier function. We hypothesized that receptor interactions with TLR2 on the epithelial surface are involved in inulin-type fructan mediated barrier modulation and that fructan chain length is also a factor which determines the effect on the epithelium. In Chapter 3 we demonstrate a model for disrupted barrier function using PMA, which causes a considerable decrease in TEER. TEER is a parameter studied as a measure for tight junction–mediated barrier function. Short- chain inulin-type fructans strongly attenuated the PMA-induced decrease in TEER, whereas long chains did not exert protective effects. These findings confirmed that chain length is a clear determinant in experimental outcome in these barrier studies. Timing also proved en essential factor in the protective capacity as no effect on recovery was observed during addition of the fructans when PMA was applied first. By blocking TLR2 with an antibody, the protective effect of short chain inulin- type fructans on barrier protection was abrogated, which confirmed the involvement of TLR2 in barrier modulation by inulin-type fructans. Thus, in addition to ligating TLR2 and modulating cytokine expression in immune

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cells, inulin-type fructans exert time-dependent and chain length– dependent protective effects on the T84 intestinal epithelial cell barrier mediated via TLR2. These results suggest that TLR2 located on both intestinal epithelial cells as well as immune cells could be a target of inulin-type fructan–mediated health effects in vivo.

Proof of principle studies confirmed direct mechanisms for inulin- type fructans in model cell types, and the established technology platform was then applied to study different fiber types. Because of renewed interest to apply fibers from chicory root byproduct (pulp) for health promoting nutrition, two major root pulp components, cellulose and pectins, were studied. In Chapter 4 the immunomodulatory and barrier modulating capacity of cellulose is described. The transcriptome of cellulose-stimulated PBMCs was studied by gene chip array and barrier function measurements were performed with human intestinal epithelial cells. Reporter assays confirmed activation through TLR/MyD88 dependent-, and independent pathways. Cellulose induced upregulation of three NF-κB related genes, i.e. cluster of differentiation 40 (CD40) molecule, interleukin 1 receptor antagonist (IL-1Ra), and interleukin-1 receptor-associated kinase 1 (IRAK1). Five upregulated genes related specifically to TLR signaling were identified, i.e. interleukin 1 receptor antagonist (IL-1Ra), interleukin-1 receptor-associated kinase 1 (IRAK1), jun proto-oncogene, mitogen-activated protein kinase kinase 3 (MAP2K3), and mitogen-activated protein kinase 13 (MAPK13). Cellulose did not improve or protect T84 resistance. From these experiments we concluded that cellulose does not directly affect intestinal cell barrier function. However, it alters gene expression in human immune cells and activates TLR and non-TLR related pattern recognition pathways, indicating the immunomodulatory potential of cellulose as major component of root pulp byproduct.

In Chapter 5 pectin, was studied for its immunogenic and barrier protective effects, as this is another major component of chicory root pulp. In addition, since pectins can differ in their chain length but also in the degree of methyl esterification, this was also studied as a structural feature, to evaluate possible structure-response properties of this molecular characteristic. Due to the lack of standardized commercially available chicory root pectin, and the similarity of the pectin profiles which can be extracted from lemon, these lemon pectins were studied as model pectins. Stimulation of TLR reporter cells with lemon pectins with different

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CHAPTER 8. GENERAL SUMMARY. degrees of methyl esterification (30DM, 56DM and 74DM), demonstrated TLR dependent activation, and an increasing TLR activation with increasing DM. The applied pectins induced TLR2 activation, and moderately activated TLR4. Upon enzymatically digesting the pectin polymers into oligomers we found that this treatment abrogated their TLR activating potential. In experiments testing the barrier function of human intestinal epithelial cell, 30DM and 74DM pectins induced a strong protective effect, while 56DM pectins induced moderate protection of T84 TEER. From these experiments we can conclude that activation of immune cells by lemon pectins is TLR2 dependent, and may also involve TLR4 ligation, and that the intact polymer backbone is indispensable for activation. Besides in activation potential, the degree of methyl esterification is also a determining factor for epithelial barrier protective effects, which were strongest for 30DM and 70DM lemon pectins.

Building on the collected in vitro results in our studies with inulin- type fructans, we hypothesized that the long chain-enriched inulin-type fructans would stimulate vaccine responses in vivo due to their predominant induction of proinflammatory cytokines, including a relatively low IL-10/IL-12 ratio, in combination with their strong ability to activate TLR2. In Chapter 6 we describe the results of a human hepatitis B vaccination and supplementation study with different inulin-type fructan supplements. By comparing the effects of long chain fructan supplementation with short chain fructans and a placebo group on anti- HBsAg titer development in the 28 days following the first injection we observed that supplementation with long chain fructans significantly enhanced the titer response (T35 of the study) as compared to the short chain supplemented group, and that a strong increased trend was present as compared to the titer development in the placebo group. Another striking observation was the identification of two responders, i.e. with a titer above 10 IU/mL plasma, in the long chain fructans group vs. no responders in either the short chain or the placebo group. Flow cytometry analysis of peripheral blood lymphocyte subsets of the supplemented individuals demonstrated that the increased titer response for the long chain group on T35 was associated with an increased percentage of TBET+ Thelper1 cells compared to basal samples, and was also significantly increased compared to all time point measurements of the placebo group. Although increased in time, at T35 the percentage of CD294+ Thelper2 cells was smallest in the long chain enriched inulin-type fructan group, compared to the short chain enriched

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inulin-type fructan group, and placebo group. These results suggest a shifted Th1/Th2 balance for the long chain group towards Th1 cell responses. Strikingly in the B cell compartment, the percentage of IgM+ cells in the non-class switched memory B cell population was decreased for the long chain group at T35 compared to basal samples. And although no significant changes were observed for the total class switched IgD- subpopulation in the long chain group and the placebo group, the relative contribution of IgG+ cells within the IgD- population in fact were increased for the long chain enriched inulin-type fructan group at time point T21 as compared to basal samples. Finally, the percentage of transitional B cells which are in the process of maturation (CD38hi IgMhi) was significantly increased in the long chain group and the placebo group for time points T14 and T21 compared to the basal samples but not in the short chain group. NK and NKT cells did not demonstrate clear supplement dependent effects, indicating that B cells and especially T cells are more involved in the boosting of the vaccination response via dietary inulin supplementation. In conclusion, this study confirms the in vivo immunostimulatory potential of long chain enriched inulin-type fructans, and subscribes an important nutritional health claim that these fibers can be beneficial for the immune system. The conclusions of this thesis are discussed in Chapter 7.

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De huidige consensus over gezonde voeding is dat voldoende dagelijkse inname van voedingsvezel kan beschermen tegen het ontwikkelen van verschillende Westerse ziekten. De precieze mechanismen waardoor deze bescherming gemedieerd wordt, zijn op dit moment nog onbekend, maar de effecten op het immuunsysteem lijken hierbij een rol te spelen. Als belangrijk onderdeel van het lichaam is het immuunsysteem verantwoordelijk voor homeostase, door het lichaam te beschermen tegen pathogenen. Bovendien is het belast met de taak om juist de commensale micro-organismen in onze darmen te tolereren, omdat deze een belangrijk onderdeel van ons verteringsstelsel vormen. De darmen worden wel gezien als het belangrijkste immunologische orgaan van het lichaam omdat ca. 70 procent van alle immuuncellen van het lichaam zich in de darm bevinden. Het darm immuunsysteem is constant actief bezig de inhoud van de darmen te ‘samplen’ om de status van het darm lumen te monitoren en goede signalen in de vorm van commensale microbiota soorten te onderscheiden van schadelijke pathogene micro-organismen en toxinen. Naast deze continue surveillance door gespecialiseerde immuuncellen, spelen ook epitheelcellen een belangrijke rol in darm homeostase. Door de sterke ononderbroken laag van epitheelcellen wordt voorkomen dat het externe milieu (waar het darm lumen ook onderdeel van is), kan doordringen in het omliggende weefsel van de lamina propria. Wanneer de darminhoud aanhoudend naar het omliggende weefsel kan lekken en ook in de rest van het lichaam terecht kan komen vanwege een defecte darmepitheel laag, noemt men dit het ‘leaky gut syndroom’. Het ontstaan van verscheidene ziekten is reeds gelinkt aan dit ongewenste fenomeen en de ontdekking van betrokkenheid in de etiologie van nog meer soorten ziekten ligt in de lijn der verwachting. Dit benadrukt het belang van een goede darmbarrière in de preventie van ziekten. Functional foods zoals prebiotische voedingsvezels hebben als doel om een gezonde darm microbiota te bevorderen door selectief de groei en activiteit van lactobacillen en bifidobacteriën te stimuleren, wat op hun beurt weer de gezondheid van de gastheer bevordert. Naast de indirecte gezondheidseffecten via bacteriën en hun korte keten vetzuur fermentatieproducten stellen wij dat er een derde mechanisme is waardoor voedingsvezels immuunmodulatie kunnen bewerkstelligen, namelijk door direct membraan receptoren op immuuncellen en epitheelcellen te activeren. Ondanks dat er een collectief gezondheids bevorderend effect wordt beschreven in de literatuur, zijn de mechanismen waardoor individuele vezelsoorten hun gezondheidseffecten induceren nog onbekend. De studies beschreven in

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NEDERLANDSE SAMENVATTING. dit proefschrift zijn er met name op gericht om directe effecten van verschillende voedingsvezeltypen uit cichoreiwortel te identificeren en te karakteriseren.

Cichoreiwortel staat bekend om het hoge gehalte van inuline-type fructanen, een van de meest bekende en bestudeerde soorten van prebiotische vezels. In Hoofdstuk 1 wordt een literatuur overzicht gegeven van de huidige kennis van immuun gemedieerde gezondheidseffecten door inuline-type fructanen. De onderzoeken waarbij deze vezels werden toegepast in de context van colon kanker, chronische ontstekingsziekten, vaccinatie studies, en infectie-, en allergie preventie, hebben geleid tot een grote verzameling wetenschappelijke bewijzen van immuunmodulerende effecten. Deze fructanen zijn hierdoor voedselcomponenten die veelbelovend zijn in de preventie van ziekten. Bij toekomstige studies over de immunologische effecten van inuline-type fructanen en andere voedingsvezels wordt aanbevolen om de keuze van immuunmarkers te correleren aan de specifieke aandoening die onderzocht wordt, en dat de relevante klinische eindpunten duidelijk worden beschreven. Bij patiëntenstudies is het noodzakelijk om aan te geven of de immuunmarkers verschillend tot expressie worden gebracht in de patiëntenpopulatie ten opzichte van de controlegroepen. Vanwege het huidige gebrek aan wetenschappelijk bewijs voor directe effecten is onderzoek nodig waarbij wordt aangetoond dat inuline-type fructanen en andere voedingsvezels directe immuunmodulatie kunnen veroorzaken door activatie van dendritische cellen (DCs) of andere immuuncellen in de darm. De hypothese van dit proefschrift luidt dan ook ”Door receptoren van het aangeboren immuunsysteem zoals Toll-like receptoren (TLRs) en Nucleotide-binding oligomerization domain-containing proteins (NODs) te activeren kan inname van voedingsvezels substantiële effecten hebben op zowel het darm immuunsysteem als immuuncellen in de periferie. Daarnaast kunnen voedingsvezels de darmbarrière beïnvloeden door activatie van dezelfde receptoren die zich op het darmepitheel bevinden”.

In Hoofdstuk 2 beschrijven we dat inuline-type fructans voor directe activatie kunnen zorgen bij contact met immuuncellen en behandelen we de rol die TLRs en NODs spelen in dit proces. Humane mononucleaire cellen uit perifeer bloed (PBMCs) werden bestudeerd als model immuuncellen, omdat deze cellen veel PRRs tot expressie brengen, zoals TLRs en NODs. Door te stimuleren met inuline-type fructanen werd de cytokine productie in PBMCs gestimuleerd. Naast dit bewijs voor

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NEDERLANDSE SAMENVATTING. directe activatie, bleken de patronen van cytokine productie ook afhankelijk van de gebruikte dosis, en bovendien van de gemiddelde ketenlengte van de fructanen. Een opvallend resultaat was dat korte keten-verrijkte inuline een regulatoire cytokinebalans induceerden vergeleken met lange keten verrijkte inuline, uitgedrukt in een respectievelijk hoge vs. lagere IL-10/IL12 ratio. Experimenten met TLR reporter cellen lieten zien dat fructan- gemedieerde activatie sterk afhankelijk is van TLRs en het TLR adapter molecuul Myeloid differentiation primary response 88 (MyD88). Uit vervolgexperimenten met reporter cellen die slechts één type TLR per cellijn tot overexpressie brengen, kunnen we concluderen dat TLR2 sterk geactiveerd werd, maar dat TLR4, 5, 7, 8, en NOD2 slechts in kleine mate geactiveerd werden. Deze resultaten bevestigen dat inuline-type fructans inderdaad directe activatie kunnen veroorzaken in humane immuuncellen, en dat deze activatie afhankelijk is van dosis, ketenlengte en voornamelijk verloopt via TLR2.TLR2 is een belangrijke receptor die betrokken is bij homeostase van de darmbarrière. De waarneming dat van alle TLRs, TLR2 voornamelijk betrokken is in inuline-type fructan herkenning leidde ertoe te onderzoeken of deze vezels de darmbarrière verbeteren of beschermen. De hypothese voor dit onderzoek hield in dat receptor interacties met TLR2 op de oppervlakte van darm epitheelcellen betrokken zijn bij darmbarrière modulatie door inuline-type fructans en dat ketenlengte ook in dit proces een rol speelt in de uitkomst van epitheel stimulatie. Tight junctions zijn verbindingspunten tussen epitheelcellen die de barrière waarborgen. TEER is een parameter die vaak wordt gebruikt om de tight junction-gemedieerde barrière functie te bestuderen. In Hoofdstuk 3 wordt een model voor schade aan de darmbarrière geïntroduceerd dat gebaseerd is op het gebruik van PMA, wat een aanzienlijke daling in de weerstand van de cellen (TEER) induceert. Uit de resultaten van deze experimenten is gebleken dat korte keten inuline-type fructanen grotendeels beschermen tegen de schade die door PMA aan de barrière veroorzaakt wordt. In tegenstelling tot de korte ketens werd dit effect niet waargenomen na behandeling met lange keten inuline-type fructans. Deze resultaten bevestigen dat ketenlengte sterk bepalend is voor de uitkomst van deze barrière experimenten. De volgorde van incubatie bleek ook een essentiële factor in het beschermende vermogen, omdat geen stimulerende effecten op herstel werden waargenomen wanneer PMA werd toegediend vóór de stimulatie met inuline-type fructanen. Door TLR2 te blokkeren met behulp van een antilichaam werd het beschermende effect van korte keten inuline-type fructanen teniet

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NEDERLANDSE SAMENVATTING. gedaan, wat bevestigt dat TLR2 betrokken is bij inuline-type fructan gemedieerde bescherming van de darmepitheel barrière. Naast activatie van TLR2 en inductie van cytokine productie in immuuncellen hebben inuline-type fructanen hier ook een beschermend effect op de barrière van humane T84 darm epitheel cellen die afhankelijk is van timing en ketenlengte en tevens gemedieerd is via TLR2.

Uit de ‘proof of principle’ experimenten kunnen we concluderen dat inuline-type fructanen directe activatie van immuuncellen veroorzaken en darmepitheel kunnen beschermen tegen schade. Het ontwikkelde technologieplatform uit deze studies werd vervolgens gebruikt om meerdere vezeltypen te bestuderen. Vanwege hernieuwde interesse in het gebruik van restproducten (pulp), die geproduceerd worden bij de extractie van inuline uit cichoreiwortel, voor gezondheidsbevorderende doeleinden, werden twee hoofdbestanddelen van deze pulp bestudeerd, namelijk cellulose en pectines. In Hoofdstuk 4 worden de immuunmodulerende effecten van cellulose beschreven. TLR reporter assays bevestigden dat cellulose immuuncellen kan activeren middels TLR/MyD88- afhankelijke en onafhankelijke pathways. Het transcriptoom van PBMCs die gestimuleerd werden met cellulose werd onderzocht d.m.v. genchip array. Uit deze experimenten bleek dat drie NF- κB gerelateerde genen opgereguleerd werden, nl. het cluster of differentiation 40 (CD40) molecuul, interleukin 1 receptor antagonist (IL- 1Ra), en interleukin-1 receptor-associated kinase 1 (IRAK1). Vijf genen die specifiek betrokken zijn bij TLR signalering werden opgereguleerd, nl. interleukin 1 receptor antagonist (IL-1Ra), interleukin-1 receptor- associated kinase 1 (IRAK1), jun proto-oncogene, mitogen-activated protein kinase kinase 3 (MAP2K3), en mitogen-activated protein kinase 13 (MAPK13). Bij bestudering van barrière effecten bleek cellulose geen bescherming te bieden tegen PMA-geinduceerde schade aan T84 darmepitheelcellen. Aan de hand van deze experimenten kunnen we concluderen dat cellulose geen directe effecten heeft op de barrière functie van darmepitheel cellen, maar cellulose induceert wel veranderingen in genexpressie in humane immuuncellen en activeert TLR- afhankelijke en onafhankelijke pattern recognition pathways, wat veelbelovend is voor het gebruik van cellulose uit cichoreipulp voor verdere experimenten.

In Hoofdstuk 5 behandelen we de immunogene en barrière beschermende effecten van een ander hoofdbestanddeel van

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NEDERLANDSE SAMENVATTING. cichoreipulp, pectine. Bovendien werden de effecten van verschillende percentages van methyl esterificatie van pectines bestudeerd, om te onderzoeken of dit structurele kenmerk van pectines ook van invloed is op de effecten die deze vezels induceren. Vanwege het feit dat cichoreiwortel nog niet opgezuiverd commercieel verkrijgbaar is, en omdat het pectine profiel wat geëxtraheerd kan worden uit citroenen sterke gelijkenis vertoont met dat van cichoreiwortel werden voor dit doeleinde pectines uit citroen gebruikt als modelpectines. Stimulatie van TLR reporter cellen met citroen pectines van verschillende graad van methyl esterificatie (30DM, 56DM en 74DM), liet een TLR-afhankelijke activatie zien, waarbij activatie toenam met toenemend gehalte aan methylgroepen. Deze pectines activeerden TLR2, en daarnaast vertoonde ook TLR4 een matige activatie. 30DM en 74DM pectines hadden daarnaast een sterke beschermende werking tegen PMA, terwijl 56DM slechts een gematigde bescherming van TEER liet zien in T84 cellen. Om te bestuderen of kleinere onderdelen van de pectines die in de darm kunnen ontstaan door vertering door bacteriën dezelfde of andere effecten hebben op TLR activatie werden de pectine polymeren enzymatisch behandeld wat leidde tot afbraak naar oligomeren. Deze oligomeren lieten echter geen enkele activatie van de TLRs meer zien. Samenvattend kunnen we uit deze experimenten concluderen dat activatie van immuuncellen DM afhankelijk is en via TLR2 verloopt, en mogelijkerwijs ook via TLR4, en dat de intacte polymeerstructuur essentieel is voor TLR activatie. Daarnaast is de graad van methyl esterificatie een bepalende factor voor de bescherming van de epitheel cel barrière.

Voortbouwend op de resultaten die verkregen zijn in de in vitro experimenten met inuline-type fructanen, hebben we de hypothese opgesteld dat lange keten verrijkte inuline-type fructanen het immuunsysteem in vivo kunnen stimuleren, vanwege het overwegend pro-inflammatoire cytokine profiel dat geïnduceerd werd, inclusief een relatief lage IL-10/IL-12 ratio, gecombineerd met een sterk vermogen om TLR2 te activeren. In Hoofdstuk 6 worden de resultaten beschreven van een humane studie naar hepatitis B vaccinatie effectiviteit in jongvolwassenen die rondom vaccinatie gesupplementeerd werden met verschillende inuline-type fructans. De effecten van lange keten fructan supplementen op de anti-HBsAg titer ontwikkeling in the 28 dagen na de eerste injectie van een hepatitis B vaccinatie werden vergeleken met de effecten van korte keten fructan supplementen en een placebogroep. Het

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NEDERLANDSE SAMENVATTING. lange keten supplement liet op T35 een significante verhoging van de titer respons zien vergeleken met de respons van de korte keten groep. Daarnaast was er een sterke, verhoogde trend zichtbaar ten opzichte van de titer ontwikkeling in de placebo groep. Een treffende waarneming daarnaast was de identificatie van twee responders, d.w.z. proefpersonen met een titer boven 10 IU/mL plasma, in de lange keten groep terwijl in de korte keten groep en placebo groep geen responders werden gevonden. Flow cytometrie analyse van lymfocytenpopulaties in perifeer bloed van de geïncludeerde proefpersonen liet zien dat de toename in titer respons in de lange keten groep op T35 gecorreleerd was met een toename in percentage van TBET+ Thelper1 (Th1) cellen vergeleken met de basaalmonsters, en tevens ten opzichte van alle gemeten tijdstippen in de placebo groep. Ondanks een algemene toename in de tijd, was het percentage CD294+ Thelper2 (Th2) cellen op T35 het kleinst in de lange keten groep vergeleken met de korte keten en de placebo groep. Deze resultaten lijken erop te wijzen dat er een verschuiving in de Th1/Th2 balans heeft plaatsgevonden in de lange keten groep richting Th1 cel responsen. In het B cel compartiment, was het relatieve aandeel van IgG+ cellen binnen de class-switched (IgD-) B cel populatie op tijdstip T21 verhoogd in de lange keten groep. Bovendien was het percentage van IgM+ cellen in de non-class switched memory B cel populatie op T35 significant verlaagd in deze groep vergeleken met de basaalmonsters. Daarnaast was ook het percentage transitionele B cellen (in het proces van maturatie, CD38hi IgMhi) significant verhoogd in de lange keten groep en de placebo groep op T14 en T21 vergeleken met de basaalmonsters maar dit was niet het geval in de korte keten groep. NK en NKT cellen lieten geen duidelijke supplement afhankelijke verschillen zien, wat erop duidt dat specifiek de B-, en T cellen betrokken zijn bij het boosten van de vaccinatie respons d.m.v. inuline supplementen. Deze studie bevestigt de in vivo immuun stimulerende functie van lange keten inuline-type fructanen en onderbouwt daarmee een belangrijke gezondheidsclaim. De bevindingen in dit proefschrift worden tot slot bediscussieerd in Hoofdstuk 7.

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Promoveren is niet gemakkelijk, en een aio kan alle hulp gebruiken die hij/zij maar kan krijgen. Als klein meisje schijn ik de eigenwijze uitspraak “zelf doen!” nogal vaak gebezigd te hebben. Inmiddels weet ik hoe fijn het kan zijn om te weten dat er mensen voor je klaar staan en dat je met elkaar veel meer kunt bereiken. Zonder begeleiders, collega’s, vrienden en familie had ik dit niet voor elkaar gebokst, dus bij deze wil ik iedereen ontzettend bedanken voor alle hulp tijdens mijn promotietraject en voor de bijdragen aan dit proefschrift!

Allereerst wil ik mijn promotor Prof. dr. P. de Vos, en mijn copromotor Dr. M.M. Faas ontzettend bedanken voor alle tijd, energie, enthousiasme en geduld die zij in dit project en de begeleiding van mijn promotietraject hebben gestoken.

Beste Paul, ik ben erg dankbaar dat jij de afgelopen 4 à 5 jaar mijn begeleider bent geweest. Je hebt me een hoop geleerd, onder andere om niet te lang in mijn eentje aan te modderen met problemen of met schrijven. De eerste versie van een stuk is immers toch nooit perfect! Door jouw “pingpong” manier van snel heen en weer sturen van een manuscript, was het meestal snel klaar om te submitten voor publicatie. Ik weet dat de drive en het ‘achter de broek zitten’ bij jou vooral voortkomt uit je enthousiasme voor het werk en je nieuwsgierigheid naar de resultaten (vaak vers van de pers, heel vaak nog in het pers-proces). Als promovendus is het gewoon erg fijn om te weten dat je begeleider zo geïnteresseerd en betrokken is bij je project. Het leek er lange tijd op dat de humane studie niet afgemaakt zou kunnen worden omdat mijn aio termijn bijna ten einde liep, maar dankzij jouw lobby voor verlenging hebben we het toch nog kunnen voltooien met mooie resultaten. Paul, heel erg bedankt voor alles!

Beste Marijke, na bij jou een leuk afstudeerproject gedaan te hebben, boden jij en Paul mij de kans om bij jullie als promovendus aan de slag te gaan. Ik ben erg dankbaar dat ik die kans gekregen heb. Wat ik in deze periode vooral van jou geleerd heb, ook naast het niet te lang zelf blindstaren op problemen, is goed kritisch te zijn op je eigen resultaten. Vooral jouw expertise op het gebied van flow cytometrie was erg welkom bij het opzetten, uitvoeren en analyseren van de humane studie. Je hebt me ook laten inzien dat een goede uitleg en weergave van de resultaten van een experiment onmisbaar zijn om begrijpelijk te zijn voor degenen die het experiment niet zelf gedaan hebben.

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Graag wil ik ook de leden van de leescommissie bedanken, Prof. dr. H.A. Schols, Prof. dr. L. Dijkhuizen, en Prof. dr. ing. W. van den Ende. Bedankt dat u in de commissie heeft willen plaatsnemen en voor het beoordelen van de wetenschappelijke kwaliteit van mijn proefschrift.

Bart, bedankt voor al je hulp op het lab, en voor het meedenken met de technische problemen die af en toe opdoken in mijn project. Vaak wisten we in de werkbesprekingen dan wel weer tot een oplossing te komen waardoor ik weer verder kon. Met nadruk bij de flow cytometrie cursus en de humane studie was jouw hulp onmisbaar. Daarnaast doen een goeie peptalk en een glaasje water van je collega(’s) wonderen als het allemaal even niet wil lukken. Gelukkig waren er ook regelmatig Nespresso pauzes, en de spinninglessen met Floor en jou waren altijd een leuke en gezellige manier om even stoom af te blazen.

Theo, jou wil ik ook heel graag bedanken voor de hulp bij de humane studie. Het is niet het meest enerverende werk geweest, maar mede dankzij jouw inzet en de honderden facsbuisjes die je hebt doorgemeten, hebben we het tot een goed einde kunnen brengen en hebben we alle samples van de proefpersonen kunnen analyseren.

Mark Boekschoten en Philip de Groot van de Universiteit in Wageningen wil ik bedanken voor de snelle en adequate analyse van de genexpressie data van de met cellulose gestimuleerde cellen.

Ik wil ook mijn aio/postdoc collega’s van ImmuunEndocrinologie bedanken voor hun hulp en natuurlijk de gezelligheid. Wat een fijne club mensen om mee samen te werken! Floor, we zijn samen in 2010 tegelijk begonnen aan het aio avontuur, en het was fijn om iemand te hebben om samen mee ‘op te lopen’, en elkaar te kunnen helpen. Mooi om te horen dat het goed gaat in Canada, je weblogs zijn altijd leuk om te lezen! Maaike, bedankt voor je hulp aan ons als opstartende aio’s, het was fijn om iemand in de buurt te hebben die al wat verder in het onderzoekstraject was en het klappen van de zweep kende. Swapnil, it took some getting used to your direct way of speaking, but you were a nice colleague to have worked with, with your own unique humor. I hope you are doing well back in India, all the best to you. Eelke, jij was een goeie toevoeging aan de groep, met leuke intitiatieven als de vrimibo en Fruity Friday. Met jouw kritische blik hebben we het voorwerk en het begin van de humane studie goed kunnen uitvoeren. Ik had het graag samen met jou afgemaakt en vond het

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DANKWOORD erg jammer dat dat er niet inzat, maar ik ben blij dat je een andere leuke baan hebt gevonden waar je in je element bent. Genaro, volgens mij heb je inmiddels het Nederlands aardig onder de knie dus dit moet wel lukken :-). Ik leerde je kennen toen we beide met dezelfde cellijnen aan het werk gingen in de celkweek ruimte, en kon het meteen erg goed met je vinden. Je bent een fijne collega, die voor andere mensen klaar staat. Ook heb ik vreselijk met je gelachen tijdens de werkborrels, dat was altijd met zere wangen van het lachen weer naar huis, geweldig. Marlies, Sandra en Gea, bedankt voor alle hulp en gezelligheid op de kamer en daarbuiten! Anne Marijn en Tsjitske, jullie wil ik ook bedanken voor alle hulp bij het werk en voor de gezelligheid. Anne Marijn, toen de humane studie zeer snel volstroomde en ik omhoog zat om “prikkers” voor alle bloedafnames heb je zelf een hoop afnames gedaan en andere collega’s weten te strikken zodat alle samples afgenomen konden worden, een reddende engel! Ik denk met plezier terug aan alle pauzes met bakjes koffie/thee/lekkere baksels/etc. Marlies, ik wil jou ook met nadruk bedanken voor je hulp bij de flow cytometrie en de humane studie. Toen daar de nood hoog was om de panels op tijd af te krijgen heb jij me echt uit de brand geholpen. Neha, I’ve gotten to know you as a hard working and very smart colleague. I enjoyed talking with you about the fibers we were working with, and discussing how they might interact with the cells we were applying them to, or just simply chatting away during our work in the cell culture room. I was honored by your invitation to attend your wedding in India, it was an amazing experience, thank you so much for letting me be a part of this milestone in your life. I would also like to thank you and Marlies for being my paranimfs, it is nice to be able to share this milestone with you! Ook zou ik mijn andere collega’s van de ImmuunEndocrinologie groep van de afgelopen 4/5 jaar willen bedanken voor het meedenken tijdens de maandagochtend werkbesprekingen: Milica, Bart Groen, Hamide, Kim, Jitty, Miriam, Floris, Alberto, en alle studenten, Eva in het bijzonder voor je hulp tijdens de cursus flow cytometrie.

De andere collega’s van de Medische Biologie wil ik ook graag bedanken voor hun hulp tijdens mijn project. De collega’s van de flow cytometrie unit, Geert, Roelof Jan en Henk, jullie wil ik erg bedanken voor de hulp bij het facsen. Met name tijdens de flow cytometrie cursus en de humane studie was die hulp zeer welkom! Ook wil ik Jelleke, Henk, en Anita bedanken voor de hulp bij het celkweken, bestellingen en andere vragen omtrent het lab. De overige collega’s van het U-lab, het Z-lab en het Allergologie lab wil ik bedanken voor hun hulp, het meedenken tijdens de

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DANKWOORD werkbesprekingen en gewoon voor de leuke werksfeer! Annet, Susan, en Hans, bedankt voor het beantwoorden van alle administratieve vragen (en hulp bij het faxen, volgens mij heb ik het nu onder de knie).

Mijn project is onderdeel geweest van een samenwerkingsverband tussen UMCG, TNO, WuR, en Cosun/Sensus (workpackage 2a) met als doel om kennisinstituten en het bedrijfsleven te laten profiteren van de kennis en technologie die in beide takken van sport aanwezig zijn. Graag wil ik Henk, Uttara, Diederick, Gerdie, Koen, Anne, en Monica bedanken voor deze fijne samenwerking, en ook voor jullie bijdrage aan de manuscripten als co-auteurs. Het is zeer leerzaam geweest om te zien hoe collega’s van verschillende achtergronden een manuscript interpreteren en jullie commentaar en suggesties zijn zeer waardevol geweest bij het schrijven van de artikelen.

Zonder proefpersonen geen humane studie. Ik wil daarom de vrijwilligers die de voedingsvezels/placebo hebben ingenomen en bloed hebben gegeven voor mijn onderzoek, heel erg bedanken. Jullie hebben een onmisbaar aandeel aan dit proefschrift geleverd. Ook de collega’s van de Transplantatie Immunologie afdeling, die vele buisjes bloed hebben afgenomen, Joan, Niels, Magdalena, Richard, en ook Laura wil ik bij deze nogmaals bedanken voor hun hulp. Van de afdeling Klinische Virologie wil ik Coretta bedanken voor het advies, en de laboranten voor het zeer snel faciliteren van nauwkeurige titerbepalingen van de hepatitis B titers.

De Biomeiden, Kitty, Inge, Mariska en Suzette. We zijn zoals de naam al doet vermoeden al vriendinnen vanaf de vroege studiejaren en ik prijs me enorm gelukkig met zo’n stel vriendinnen die elkaar door dik en dun steunen. We hebben al heel wat met elkaar meegemaakt en menige mijlpalen in elkaars leven mogen delen. Susan, lief peuterspeelzaal vriendinnetje van me, ik ben heel erg blij dat ik deze gebeurtenis ook met jou kan delen! Het was nog even de vraag of het tijd-technisch, topografie- technisch en Milou-technisch zou lukken, maar met de verlenging van mijn project komt het allemaal op zijn pootjes terecht. Arenda, door de drukte in onze beide levens zien we elkaar wat minder vaak, maar zodra we weer aan de klets zijn is het altijd erg gezellig! Ik denk met heel veel plezier terug aan de vakanties die we samen op Kos en in Portugal hebben gevierd. Lieve vriendinnen, bedankt voor al jullie steun tijdens mijn promotietraject. Ook al veranderen onze levens met verschillende woonplaatsen, drukke banen, gezinsuitbreidingen, etc.etc. toch blijft de

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DANKWOORD band met jullie voor mij bijzonder en ik wil jullie bedanken dat jullie er voor me zijn!

Pap en mam, jullie hebben het mogelijk gemaakt dat ik biologie kon gaan studeren en hebben me altijd geweldig gesteund tijdens die studie en daarna ook tijdens mijn promotietraject. Ik herinner me nog goed jullie aanmoedigingen bij het afronden van de toch wel lastige literatuur review, tijdens het weer tijdelijk thuis wonen, het relativeren, de schoppen onder de kont, het uitdokteren of ik genoeg budget had om op een gegeven moment die mooie woning van Marcel in Groningen te kunnen betrekken, het trotse gevoel wanneer ik kon vertellen dat een artikel geaccepteerd was voor publicatie, het proberen uit te leggen wat dat artikel dan eigenlijk inhield, en de zeer welkome uitjes richting het bronnenbad in Bad Nieuweschans, in de stressvolle, drukke periodes (of gewoon tussendoor). Heel erg bedankt voor alles!

Robin, Roy en Mariska, lieve broers en schoonzusje, bedankt voor alle hulp, steun, humor, grappen, grollen, geouwehoer, lachen, tranen (van het lachen meestal), ik ben blij dat ik jullie heb, ik weet dat ik altijd op jullie kan rekenen, of het nou is voor hulp bij een (zoveelste) verhuizing, wazige filosofische gesprekken over de zin van het leven om 2.30 a.m., een peptalk, kaasfonduen of andere culinaire bezigheden, gezellig borrelen, het is heel fijn om zo’n goeie band met elkaar te hebben, dankjulliewel!

En opeens heb je dan wat (schoon) familieleden erbij! Anneke, Jorien, Rinske, Pascal, Jan en Anita, de Vernimmentjes/Oddinkjes, wat leuk dat ik jullie heb mogen leren kennen! Gezellig samen eten, weekendjes in het Waddengebied, leren Carioca te spelen, Sinterklaas vieren, één en al gezelligheid! En een hele goeie afleiding van de drukte van het werk. Rinske wil ik in het bijzonder nog even een keertje bedanken voor haar inzet voor de humane studie, door deelname en het voorstellen van kandidaat proefpersonen is de studie volgekomen, dankjewel!

En als laatste, Yke. Op een laatste plaats genoemd worden, wil in de geschreven wetenschap meestal zeggen dat je van essentieel belang bent geweest voor het manuscript. Lieve Yke, ik kan je niet genoeg bedanken! (maar ik ga het toch proberen). Toen we elkaar leerden kennen, vertelde je dat je zo’n 250 km verderop woonde, in Rotterdam. Na een tijdje daten in Groningen werd het wel eens tijd dat ik die kant op ging. Daar heb je me

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DANKWOORD laten zien hoe mooi en gezellig die stad is en heb je me voorgesteld aan al die gekke, lieve vrienden van je. - Hen wil ik eigenlijk ook nog even bedanken voor alle gezellige activiteiten in het Rotterdamse, de speciaalbiertjes/wijntjes/andere drankjes, de pubquiz, de barbecue in het Vroesenpark, en zelfs het vrijgezellenfeest, en de bruiloft van Sarah en Olaf, heel bijzonder om dat te mogen bijwonen, dankjulliewel! - Yke, we hebben elkaar leren kennen in het laatste jaar van mijn promotietraject, in een periode die, naarmate de tijd vorderde, eigenlijk alleen maar drukker werd. Rotterdam was niet naast de deur, maar toch hebben we samen die afstand weten te overbruggen en ben je er altijd voor me geweest. Zelfs toen ik eigenlijk veel te weinig tijd vrij kon maken voor jou heb je altijd geduld gehad en me gesteund met bakjes Ykiaanse koffie, anti-stress schoudermassages, opbeurende woorden en afleiding waar nodig. Jouw liefde en warmte zijn een enorme steun in de rug geweest en ik ben ontzettend dankbaar dat we elkaar hebben gevonden! Ik kijk er erg naar uit om met jou te kunnen gaan samenwonen en mijn leven nog meer met jou te kunnen delen, ik hou héul véul van je!

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Publication list

Vogt L.M., Boekschoten M.V., de Groot P.J., Faas, M.M., de Vos P. Cellulose alters the expression of nuclear factor kappa B-related genes and Toll-like receptor-related genes in human peripheral blood monocytes. Journal of Functional Foods 2015 (in press).

Vogt L.M., Meyer D., Pullens G., Faas M.M., Smelt M.J., Venema K., Ramasamy U., Schols H.A., de Vos P. Immunological properties of inulin- type fructans. Critical Reviews in Food Science and Nutrition 2015. Vol. 55, p. 414-436.

Vogt L.M., Meyer D., Pullens G., Faas M.M., Venema K., Ramasamy U., Schols H.A., de Vos P. Toll-like receptor 2 activation by β2→1 fructans protects barrier function of T84 human intestinal epithelial cells in chain length-dependent manner. Journal of Nutrition 2014. Vol. 144, p.1002- 1008.

Vogt L.M., Ramasamy U., Meyer D., Pullens G., Venema K., Faas M.M., Schols H.A., de Vos P. Immune modulation by different types of β2→1- fructans is Toll-like receptor dependent. PLOS ONE 2013. Vol. 8, p. e68367.

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Curriculum Vitae

Leonie Marloes Vogt was born in Delfzijl, The Netherlands, on the 18th of August 1983. In 2001 she graduated from high school (Dollard College) and started her studies in Biology at the University of Groningen. She obtained her Bachelor’s degree with the specialisation Medical Biology in 2004. During her Masters in Biomedical Sciences she performed her first research project at the Cell Biology Department of the University Medical Center Groningen (UMCG) studying the impact of different extracellular matrix proteins on the remyelination capacity of oligodendrocytes, which play an important role in multiple sclerosis. Her second research project was performed at the Medical Biology division of the UMCG, during which she studied the molecular pathways which are induced in immortalized B cells by Rituximab, a therapeutic which is applied in the treatment of rheumatoid arthritis. Her final Master project was performed in the group of Dr. Marijke Faas and Prof. dr. Paul de Vos, studying the differences in adhesion to and activation of monocytes and endothelial cells by syncytiotrophoblast microparticles (STBM) of healhy pregnant versus preeclamptic women. After finishing with a colloquium on the potential of cigarette smoke components in the treatment of inflammatory bowel diseases, she obtained her Master degree in winter 2009. After a three month interim position as a teacher of Physics at the Fivel College in Delfzijl, she started a PhD project on dietary fibers and intestinal health, in the Medical Biology division of the UMCG under supervision of Prof. dr. Paul de Vos. This project was performed in collaboration with Wageningen University, TNO, and Cosun/Sensus, as a workpackage within the Carbohydrate Competence Center.

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