Immune-modulatory effects of dietary Yeast Beta-1,3/1,6-D-glucan

Introduction

A well-functioning immune system is crucial for staying healthy. Therefore, the potential of natural substances to strengthen the immune system has long been the subject of investigation. There are many synthetic and natural preparations claiming to be immunomodulators. Probably the best known herbal preparations that exhibit effects on the immune system are preparations made from Echinacea, Viscum (mistletoe), and Pelargonium. There is, however, another very interesting and, by now, properly investigated class of immunomodulators - the β-glucans.

Long before the substance class of β-glucans themselves were identified as immunomodulators, the beneficial effects of β-glucan-containing mushrooms such as Shiitake (Lentinus edodes) in Japan or Lingzhi (Ganoderma lucidum) in China were utilized in the traditional Oriental medicine for the strengthening of the body’s immune system.

The research on β-glucans started in the middle of the last century, when the immune-modulating effect of a yeast insoluble fraction was first shown [1]. Later it was demonstrated that the immunological activity of this preparation derives from the β-(1,3)-D-glucans [2].

Most of our current knowledge on the health benefits of β-glucans, the underlying mode of action, and its relationship to the structure of β-glucans was discovered within the last 20 years (for review see [3,4]).

Meanwhile, more than 6000 publications investigating the immune-modulating effects of β-glucans, such as anti-inflammatory or antimicrobial abilities, have been published [5]. Health effects were found not only in humans but also in invertebrates, rodents, fishes as well as farm animals such as cows or pigs (for review see [4]). Further, numerous studies reported other health benefits of β-glucans, including hepatoprotective, wound healing, weight loss, antidiabetic and cholesterol lowering functions (for review see [6,7]).

β-glucans are a heterogeneous group of natural polysaccharides, consisting of D-glucose monomers linked by a β-glycosidic bond. They are important structural elements of the cell wall or serve as energy storage in bacteria, fungi including yeast, algae, and plants, while they are absent in vertebrate and invertebrate tissue.

The individual glucose subunits are primarily linked either by (1,3)-β, (1,4)-β, or (1,6)-β glycosidic bonds. In most cases, β-glucans exhibit a uniformly constructed backbone of various lengths with side-chains of D-glucose attached by (1,4)-β, or (1,6)-β bindings.

However, not all β-glucans are able to modulate immune functions. These properties mainly depend on the primary chemical structure of the β-glucans. Cellulose for example, a (1,4)-β-linked glucan, does not exhibit immune-modulatory effects. In contrast, β-glucans derived from fungi and yeast, which consist of a (1,3)-β-linked backbone with small numbers of (1,6)-β-linked side chains, are essentially known for their immune-modulating effects [8].

Apart from differences in the type of linkage and branching, β-glucans can also vary in solubility, molecular mass, tertiary structure, degree of branching, polymer charge and solution conformation (triple or single helix or random coil). All these characteristics may influence their immune modulating effects [9]. On the other hand, the manufacturing process and, hence, the isolation method impacts the structure of β-glucans and consequentially their effects on the immune system. Indeed, the immune-modulating activity of different β-glucans from the same source might differ considerably in level of purity, solubility, molecular mass, tertiary structure, degree of branching, polymer charge and solution conformation [5].

Although there are numerous in vitro and in vivo investigations, human clinical trials confirming the preclinical findings are rather scarce. Due to different natural sources of the β-glucans, differences in application (intraperitoneal, intravenous or subcutaneous injections, or oral), differences in preparation and thus structural differences, the results obtained in the resulting clinical studies are non-homogeneous and often contradictory [4].

Due to this heterogeneity, we focused our review on the immune-modulating effects of insoluble yeast-derived dietary β-glucans.

The main focus will be on the food product Yestimun® - an insoluble, highly purified, well-characterized and intensively studied β-glucan from Spent Brewers’ Yeast. The in vivo data obtained from this preparation, together with the efficacy results obtained during clinical trials, will be presented and compared to the other dietary, insoluble yeast β-glucan preparations available on the market. Only randomized, controlled trials were considered for the purpose of this review. Possible mechanisms of action will be presented, based on the current knowledge about structure-function relationships of β-glucans. Studies investigating other aspects than the immune system or studies with soluble β-glucans were excluded.

Characteristics of the proprietary yeast β-glucan preparation (Yestimun®)

Yestimun® is an insoluble (1,3)-(1,6)-β-glucan made from Spent Brewers’ Yeast (Saccharomyces cerevisiae). The brewers’ yeast used in Yestimun® is grown exclusively on malt and clean spring water with no other nutrients added. It is a natural by-product of the fermentation process used for beer production. Following gentle autolysis with the yeast’s own enzymes, high-performance centrifuges are used to separate the yeast autolysate into the soluble yeast extract and the insoluble yeast cell wall. Yeast cell walls contain typically about 30% of β-glucans of dry weight. During several separation processes, the β-glucans are further purified without the use of strong alkaline in the hydrolysis process, and soluble compounds are removed. Furthermore, the process does not involve an acidic hydrolysis, leaving the acid-sensitive β-1,6-glucan side chains mainly intact. This results in an average of 22% relative linkage percentage from β-1,6 glucan (H-NMR analysis according to FCC VII, 3rd suppl.; reference-glucan at 14%), with a minimum of purity of 85% (dry mass).

Growth conditions of the yeast might result in some heterogeneity of β-glucans within the cell wall [4]. Therefore, it can be speculated that β-glucans from cell walls of S. cerevisiae grown during brewery have a different β-glucan pattern than those from bakers’ yeast, which might also influence the immuno-modulating abilities of the product.

Mode of action/structure function relationship

As humans cannot metabolize the β-glycosidic bonds from β-glucans, it has long been suspected that the bacterial fermentation process taking place within the intestinal system is involved in the health promoting effect of β-glucans.

Meanwhile, different possible mechanisms have been identified on how oral β-glucans modulate the immune system (for review see [10,11]).

In general, humans cannot synthesize β-glucans. Therefore, the immune system recognizes these compounds as foreign. The innate immune system responds to invading pathogens through pattern recognition receptors (PRR), which are typically expressed by immune cells but also by other cells. PRRs recognize conserved microbial structures, the so-called microbe-associated molecular patterns (MAMPs) [12], formally called PAMPs [13,14]. β-glucans are considered as one of the major MAMPs for the PRR-mediated sensing of fungal infection. So far, the most important PRRs for β-glucans are the dectin-1 receptor, the complement receptor 3 (CR3) and toll-like-receptors (TLR), which are found on various immune cells such as monocytes, macrophages, dendritic cells, neutrophils, eosinophils, and natural killer cells, but also on intestinal epithelial cells [10,15-17]. Binding of β-glucans to dectin-1 induced a cascade of innate and adaptive immune response such as phagocytosis, oxidative burst, and the production of cytokines and chemokines in dentritic cells and macrophages [15]. Kankkunen et al. showed that particulate yeast β-glucan triggered interleukin-1β (IL-1β) mediated cellular response in human primary macrophages via dectin-1 signaling [18]. Earlier in vitro studies showed that yeast β-glucan is a strong stimulant of macrophages [19] and induced mitogenic activity in rat thymocytes, indicating immunostimulatory effects [20].

The exact mechanism on how β-glucans affect immune function depends in part on the route of administration. Strong effects were already observed in studies in the early 1990s using intravenously injected yeast β-glucans [21-23], when the biological efficacy of orally administered β-glucans was critically discussed. In terms of oral administration, the impact on immune function is assumed to be primarily explained by the interaction of β-glucans with pinocytic microfold (M)-cells located in the small intestine [24]. It has been suggested that M-cells take up β-glucans and transport them from the intestinal lumen to the immune cells located within the Peyer’s patches [11].

Uptake of β-glucans has been shown in mice with soluble and particulate yeast (1,3)-(1,6)-glucans labeled with fluorescein. Both types of yeast β-1,3-glucans were taken up by gastrointestinal macrophages, and then processed forms were transported to the lymph nodes, spleen and bone marrow [25]. Also, in vitro experiments have shown that β-glucans were degraded inside macrophages and released into the culture medium [25], which makes them eventually available for the circulating system and a systemic distribution.

Orally administered β-glucans induced phagocytic activity, oxidative bursts, and IL-1 production of peritoneal macrophages in mice [26]. A higher phagocytic activity and oxidative metabolism of neutrophils and monocytes, indicating an immune restoring activity of yeast β-glucan has also been shown in rats [27]. However, not only the cellular but also the humoral acute phase immune reaction is affected by yeast β-glucan feeding as shown by increased lysozyme and ceruloplasmin activitiy [28].

Moreover, oral delivery of β-glucans impact mucosal immunity, as shown by an increase of intraepithelial lymphocytes in the intestine of mice [29]. In rats, the absorption of soluble β-glucans translocated from the gastrointestinal tract into the systemic circulation leads to an increased immune response and resistance against infectious challenge [16]. The effect of an insoluble β-glucan against anthrax infection in mice showed that the treated animals survived the anthrax infection, while 50% of the control animals died, indicating an improved immune function in animals fed with β-glucan [30].

Another important aspect to be considered is the solubility of β-glucans, as soluble and particulate (insoluble) β-glucans isolated from yeast may stimulate the immune system via different pathways [31]. In vivo and in vitro studies revealed that particulate (insoluble) β-glucan was phagocytosed by dendritic cells and macrophages via dectin-1 receptor pathway. Although particulate β-glucans can also be taken up by dendritic cells through a dectin-1 receptor independent mechanism, the dectin-1 receptor pathway is essential for the activation of dendritic cells, which in turn induces T-cell response and cytokine release [31]. However, not all insoluble particulate β-glucans are able to bind to and activate the dectin-1 receptor. Studies with synthetically produced β-glucans revealed that binding to dectin-1 receptor is specific for β-glucans with a (1,3)-beta backbone. Backbones with mixed (1,3)/(1,4)-β-bindings (e.g. barley derived β-glucans) are not recognized by this receptor. Also, a backbone length of at least seven glucose units is required for binding, in addition to one (1,6)-β-side-chain branch (e.g. insoluble yeast β-glucans). Furthermore, the binding activity increases with increasing molecular weight of the polymer [32]. However, binding to the dectin-1 receptor alone does not activate the signal cascade induced by this receptor. Indeed, dectin-1 signaling and the concomitant immune responses are only activated by particulate β-glucans but not by soluble β-glucans [33]. Insoluble, particulate β-glucans induced the process of phagocytosis, resulting in the elimination of invading microbes by binding to the dectin-1 receptor [31]. Further, particulate β-glucans promotes T-cell differentiation into Th1-cells and enhances cytotoxic T lymphocyte priming by the dectin-1 pathway. Even though soluble β-glucans are also recognized by the dectin-1 receptors, they cannot activate immune response via this pathway. Soluble β-glucans are, however, able to bind to the CR3 receptor. The activation of the CR3 leads to complement system mediated immune process, supported by specific antibodies [31].

These results show that different β-glucan particles influence the immune system via different pathways. Insoluble β-glucans are able to activate both the innate and the adaptive immune responses, whereas soluble β-glucans are most effective via the complement system, which needs specific antibodies.

Studies performed with the proprietary yeast β-glucan preparation (Yestimun®)