Structure–function relationships of immunostimulatory polysaccharides: A review

Carbohydrate Polymers 132 (2015) 378–396

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Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol

Review

Structure–function relationships of immunostimulatory polysaccharides: A review Sónia S. Ferreira a , Cláudia P. Passos a , Pedro Madureira b,c,d , Manuel Vilanova b,c,d , Manuel A. Coimbra a,∗ a

QOPNA, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal Instituto de Investigac¸ão e Inovac¸ão em Saúde, Universidade do Porto, 4200-135 Porto, Portugal c IBMC—Instituto de Biologia Molecular e Celular, Universidade do Porto, 4150-180 Porto, Portugal d ICBAS, Instituto de Ciências Biomédicas de Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal b

a r t i c l e

i n f o

Article history: Received 10 April 2015 Received in revised form 28 May 2015 Accepted 31 May 2015 Available online 9 June 2015 Keywords: Immunomodulation Triple helix Branching Acetylation Sulfation Glucans

a b s t r a c t Immunostimulatory polysaccharides are compounds capable of interacting with the immune system and enhance specific mechanisms of the host response. Glucans, mannans, pectic polysaccharides, arabinogalactans, fucoidans, galactans, hyaluronans, fructans, and xylans are polysaccharides with reported immunostimulatory activity. The structural features that have been related with such activity are the monosaccharide and glycosidic-linkage composition, conformation, molecular weight, functional groups, and branching characteristics. However, the establishment of structure–function relationships is possible only if purified and characterized polysaccharides are used and selective structural modifications performed. Aiming at contributing to the definition of the structure–function relationships necessary to design immunostimulatory polysaccharides with potential for preventive or therapeutical purposes or to be recognized as health-improving ingredients in functional foods, this review introduces basic immunological concepts required to understand the mechanisms that rule the potential claimed immunostimulatory activity of polysaccharides and critically presents a literature survey on the structural features of the polysaccharides and reported immunostimulatory activity. © 2015 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379 Basic immunological concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379 Polysaccharides structural features conferring immunological properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 Polysaccharides with reported immunostimulatory activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 4.1. Glucans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381 4.1.1. Conformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387 4.1.2. Molecular weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388 4.1.3. Functional groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388 4.1.4. Branching degree . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388 4.1.5. Synopsis of glucans immunostimulatory activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388 4.2. Mannans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388 4.2.1. Conformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388 4.2.2. Molecular weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 4.2.3. Functional groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 4.2.4. Branching degree . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389

∗ Corresponding author. Tel.: +351 234 370706; fax: +351 234 370084. E-mail address: [email protected] (M.A. Coimbra). http://dx.doi.org/10.1016/j.carbpol.2015.05.079 0144-8617/© 2015 Elsevier Ltd. All rights reserved.

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4.3.

5.

Pectic polysaccharides and arabinogalactan proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390 4.3.1. Conformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390 4.3.2. Functional groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 4.3.3. Branching degree . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 4.4. Galactans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 4.4.1. Molecular weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 4.4.2. Functional groups . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 391 4.5. Fucoidans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 4.6. Hyaluronans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 4.7. Fructans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 4.7.1. Conformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 4.7.2. Molecular weight . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 392 4.8. Xylans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393

1. Introduction According to the Scopus Database, until 2014, 9004 papers have been related with immunological activities of polysaccharides, the majority related to bacterial lipopolysaccharides, due to lipid A (Meng, Lien, & Golenbock, 2010). However, a large number of polysaccharides, namely those of plants (Xing et al., 2013), fungi (Nie, Zhang, Li, & Xie, 2013), yeasts, and algae (Campo, Kawano, Silva, & Carvalho, 2009), lacking the lipid moiety, have also been reported to interact with cells of the immune system, as well as with molecules involved in humoral immunity. These interactions have been mainly associated with potential stimulatory effects on the immune system, strengthening innate and adaptive immune responses, either by exhibiting the effect themselves or by inducing effects via complex reaction cascades (Dalmo & Bøgwald, 2008; Ramberg, Nelson, & Sinnott, 2010; Schepetkin & Quinn, 2006). The antimicrobial (Holderness et al., 2011; Skyberg et al., 2012), antiviral (Ohta et al., 2007), antitussive (Nosál’ová et al., 2011), radioprotective (Mao et al., 2005), anti-septic shock (Tzianabos, 2000), and antitumoural (Wasser, 2002; Zong, Cao, & Wang, 2012) immune-related properties of these polysaccharides make them envisaged as potential promoting or even therapeutical agents. From the chemical point of view, systematic studies providing structure–function relationships of immunostimulatory polysaccharides are generally lacking. In addition to the disperse information in this regard, whenever available, chemical characterization of the polysaccharides is often insufficient. Low purification, as well as the high heterogeneity of the polysaccharide structures involved, have prevented the establishment of broad structure–function relationships (Ramberg et al., 2010) beyond source-function relationships (Jin, Zhao, Huang, Xu, & Shang, 2012; Nie & Xie, 2011; Sun, 2011; Xu, Yan, Tang, Chen, & Zhang, 2014). Therefore, this review will attempt to contribute to the systematization of the already available information concerning the structure–function relationships of immunostimulatory polysaccharides. This is an important first step for the design of polysaccharides with immunostimulatory activity to be used in new health therapeutics and/or incorporation in functional foods, with health benefits. 2. Basic immunological concepts The immune system comprises body defences against foreign or potentially dangerous invaders, generally referred as innate or nonspecific immunity and acquired or specific immunity. Immune components include, among others, physical barriers (skin and mucosae), soluble factors mediating the so-called humoral immunity (such as antibodies and complement proteins) and

leukocyte cells (cellular immunity) which can communicate via low molecular-weight soluble proteins, generally referred to as cytokines (Paul, 2012). Innate immunity is the first line of defence. It does not require a previous encounter with pathogens or other foreign material to work effectively, providing an immediate response to invaders. Besides the skin and mucosal barriers, it includes phagocytic cells such as monocytes, macrophages, neutrophils, and dendritic cells, as well as diverse populations of innate lymphoid cells. Acquired immunity depends on B and T lymphocytes, and antigen-presenting cells. Although more specific, acquired immunity takes time to develop after the initial antigenic stimulus. However, thereafter, its response is quick. The activation of innate immune responses produces signals that stimulate and direct subsequent adaptive immune responses which in turn can amplify or down-regulate innate immune mechanisms (Fearon & Locksley, 1996). Therefore, innate and adaptive immunity operate in cooperative and interdependent ways. Immunostimulatory polysaccharides can interact direct or indirectly with the immune system, triggering several cellular/molecular events, leading to immune system activation (Leung, Liu, Koon, & Fung, 2006) (Fig. 1). Monocytes, macrophages, and neutrophils are the main targets described responding to these molecules. When searching for reported immunostimulatory activities of polysaccharides, most studies addressed macrophage function (Schepetkin & Quinn, 2006). Several in vivo studies have shown an enhanced macrophage phagocytic function induced by treatment with immunostimulatory polysaccharides, like (␤1 → 4)-d-glucans with side chains of C-3 (␤1 → 6)-d-Glc units (Zhao, Chen, Ren, Han, & Cheng, 2010), (␤1 → 4)-d-mannans (Leung et al., 2004), (␣1 → 6)-d-Galp branched (␤1 → 3)-d-mannans (Sun et al., 2008), porphyran (sulfated galactan) (Bhatia et al., 2013), and arabinoxylans (Zhou et al., 2010). Beyond phagocytosis, immunostimulatory polysaccharides may also enhance other macrophage functions, such as production of reactive oxygen species (ROS) and nitric oxide (NO) (Xie et al., 2008), secretion of pro-inflammatory cytokines, such as tumour necrosis factor (TNF-␣), interleukin (IL)-1, IL-6, IL-8, IL-12, and interferon (IFN)-␥ (Khil’chenko et al., 2011; Ohta et al., 2007; Schepetkin, Faulkner, Nelson-Overton, Wiley, & Quinn, 2005; Xu, Yan, & Zhang, 2012). Moreover, several immunostimulatory polysaccharides have also been reported to affect macrophage proliferation and differentiation (Ramesh, Yamaki, & Tsushida, 2002). In addition to inducing macrophage responses, complementfixing activity has been attributed to immunostimulatory polysaccharides, including mixed (␤1 → 4)(␤1 → 3)-d-glucans (Samuelsen, Rieder, Grimmer, Michaelsen, & Knutsen, 2011), pectic polysaccharides (Nergard et al., 2005), type II arabinogalactans (Inngjerdingen et al., 2005, 2007a), and porphyrans (Bhatia et al.,

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Fig. 1. Illustration of immune system activation by immunostimulatory polysaccharides after interaction and trigger of several molecular/cellular events. Abbreviations: AG-II, type II arabinogalactans; Akt, protein kinase B; CD, cluster of differentiation; CR3, complement receptor 3; ERK 1/2, extracellular signal regulated kinase 1/2; GSK3, glycogen synthase kinase 3-␤; H2 O2 , hydrogen peroxide; IFN, interferon; IL, interleukin; JNK 1/2, Jun N-terminal kinase 1/2; JNK, c-Jun N-terminal kinase; MAPK, p38 mitogenactivated protein kinase; MHCII, major histocompatibility complex class II; NO, nitric oxide; O2•− , superoxide anion; PAK, p21-activated kinase; PI3K, phosphatidylinositol-3 kinase; PTK, protein-tyrosine kinase; Rac 1, Ras-related C3 botulinum toxin substrate 1; SR, scavenger receptor; Src, proto-oncogene protein kinase; TLR-4, toll-like receptor 4; and TNF-␣, tumour necrosis factor ␣.

2013), which has been suggested to contribute to their stimulatory properties on immune cells. The activation of innate immune cells and of the complement system can also be involved in the activation of other cells and adaptive immunity. Nevertheless, direct activation by immunostimulatory polysaccharides of other immune cells than macrophages, like NK cells and B lymphocytes was also shown (Inngjerdingen et al., 2005, 2007a,b). The polysaccharides’ stimulatory effect on lymphocyte cells has been evaluated mainly by induction of proliferation (Du et al., 2014; Inngjerdingen et al., 2007a; Qiao et al., 2010b; Wang, Liu, & Fang, 2005; Zhao et al., 2010), and cytokine production (Holderness et al., 2011; Nosál’ová et al., 2011; Skyberg et al., 2012). Activation of the immune system by polysaccharides is thought to be mediated primarily through their recognition by specific receptors that will determine the resulting response. The so-called pattern recognition receptors (PRRs), surface or intracellularly expressed by the different immune cell types, are germlineencoded receptors that recognize conserved molecular structures (pathogen-associated molecular patterns) shared by broad groups of pathogens that are generally absent from the host. PRRs found on the cell membrane include, among others, scavenger receptors (SRs) (Canton, Neculai, & Grinstein, 2013) and toll-like receptors (TLRs) (Song & Lee, 2012). TLRs are a family of ancient PRRs that can link innate and adaptive immunity (O’Neill, Golenbock, & Bowie, 2013). SRs are present on macrophages and many types of dendritic cells, and include Class A SR, ␤-d-glucan receptor Dectin-1, mannose receptor (MR), and complement receptor type 3 (CR3). Additionally, some innate immunity receptors are present in the bloodstream and tissue fluids as serum proteins, in addition to specific antibody. In blood plasma, soluble pattern receptors include mannan-binding lectin (MBL), ficolins, C-reactive protein (CRP), lipopolysaccharide-binding protein (LBP), and proteins of the alternative and classical complement pathways (Carroll, 2004; Thiel &

Gadjeva, 2009). B-cell and T-cell antigen receptors are clonally distributed lymphocyte receptors that allow the fine-discriminative capacity of adaptive immunity by recognizing specific structural details of antigen molecules (Hsu, 2011). The interaction of immunostimulatory polysaccharides with cell receptors may trigger signalling pathways, ultimately leading to induction of gene transcription, as detailed for lentinan, a (␤1 → 3)d-glucan (Xu et al., 2014). In macrophages, binding of fucoidan to SR triggers two signalling pathways: protein-tyrosine kinase (PTK) via a proto-oncogene protein kinase (Src)/Ras-related C3 botulinum toxin substrate 1 (Rac1)/p21-activated kinase (PAK)/cJun N-terminal kinase (JNK) and PTK(Src)/Rac1/PAK/p38, playing critical roles in proIL-1/IL-1 regulation (Hsu, Chiu, Wen, Chen, & Hua, 2001). The interaction of fucoidan with SR on human peripheral blood dendritic cells increased the surface expression of maturation cell marker CD83 and of co-stimulatory and antigenpresenting molecules (CD80, CD86 and major histocompatibility complex class II). The fucoidan-induced maturation of these cells was shown to depend on the induction of TNF-␣ production, mediated by p38 mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3-␤ (Jin et al., 2009). The interaction of particle and soluble (␤1 → 3)-d-glucans with Dectin-1 and CR3, respectively, activates innate immune functions (such as oxidative burst activity) by overlapping distinct signalling pathways (Bose et al., 2014). Dectin-1-mediated activation requires Src family kinases phosphatidylinositol-3 kinase and protein kinase B/Akt. In contrast, CR3-mediated activation requires focal adhesion kinase, spleen tyrosine kinase, phosphatidylinositol-3 kinase, Akt, p38 mitogen activated protein kinase, phospholipase C and protein kinase C. The interaction of (␤1 → 4)-d-mannans with immune system can occur through binding to MR (Karaca, Sharma, & Nordgren, 1995) but also TLR4. The interaction of mannans with TLR4 in macrophages mediated activation of extracellular signal-regulated kinase (ERK) 1/2, JNK 1/2, and p38 MAPK (Liu et al., 2008). Activation of macrophages

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with polysaccharides obtained from the roots of Actinidia eriantha Benth was shown to upregulate the expression of involved in NF␬B signalling pathways, several TLR genes and genes encoding pro-inflammatory cytokines such as IL-1␤, IL-1␣, IL-6, TNF-␣, IFN␤, TNF-␤, as well as anti-inflammatory cytokine IL-l0 (Sun et al., 2015). The polysaccharide-driven activation of transcription factors may induce the expression of other inflammatory mediators than cytokines, such as inducible nitric oxide synthase (iNOS) (Luo, Xu, Yu, Yang, & Zheng, 2008; Schepetkin et al., 2005) or cyclooxygenase (COX)-2 (Sun et al., 2015). Although the majority of the studies reporting immunostimulatory activity of polysaccharides do not characterize signalling pathway details and/or identify the transcription factors involved, the responses observed must result from the activation of such pathways. Table 1 summarizes relevant assays published so far concerning the establishment of relationships between polysaccharides structural features and observed immunostimulatory activity.

3. Polysaccharides structural features conferring immunological properties The first studies that related polysaccharides to immune activity were those related with the antigenic polysaccharides from bacteria (Morgan, 1936). After these, less toxic polysaccharides from alternative sources such as plants, fungi, animals, and algae were progressively studied (Ramberg et al., 2010; Schepetkin & Quinn, 2006). Nowadays, several types of immunostimulatory polysaccharides can be found in the literature. However, the lack of sufficient isolation, purification, and structural characterization has prevented the establishment of a broader range of structure–function relationships. Several immunological studies have been done only with non-purified polysaccharide-rich extracts. This way, the presence of other compounds, like polyphenols (Ebringerová et al., 2008), proteins (Zhao et al., 2012), or contaminants like lipopolysaccharides (LPS) (Schepetkin & Quinn, 2006), could affect the measured activity. The presence of mixtures of different polysaccharides in the same sample can also mask or interfere with the immunostimulatory activity of individual components (Smiderle et al., 2011, 2013). In such cases, fractionation methodologies and chemical characterization (Yang & Zhang, 2009) are important steps for purification and identification of true structure–function relationships. Glucans, mannans, pectic polysaccharides, arabinogalactans, fucoidans, galactans, hyaluronans, fructans, and xylans are the most studied polysaccharides concerning their possible immunostimulatory activity (Fig. 2a). However, only 12% of the searched papers in Scopus Database define the type of polysaccharide under study. From these reports, the majority do not associate any structural feature to the reported immunostimulatory activities, showing that it is still unclear how a defined structure may contribute to these activities. Also, there is some misleading nomenclature concerning the origin of type II arabinogalactans, as polysaccharides with similar structures may be present as pectic polysaccharides side chains and as components of arabinogalactan proteins (AGP). In addition to the type of polysaccharide, conformation, molecular weight, presence of functional groups like acetyl and sulfate groups, and branching degree are connected with the immune topic (Fig. 2b). These structural features may have different relevance depending on the type of polysaccharides and their combination may impact the resulting immunostimulatory activity in different ways. Polysaccharide conformation is related to the spatial arrangement of the atoms that determine the shape adopted by the chain molecule (Preston, 1979). It depends on the type of linkage between the sugar units of the chain, presence of branches, and neighbouring molecules. The polysaccharides heterogeneity, their high

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molecular weight, and the complexity of some structures, promoting the formation of aggregates in solution, can mask the behaviour of the individual molecules and difficult the analysis of their conformation (Yin et al., 2012). Polysaccharides may exhibit different conformations in solution, such as helical, including single- and triple-helix, and random coil, that can be more or less stiff (Yang & Zhang, 2009). This structural characteristic can influence the direct contact between the polysaccharides and the cells or other components of the immune system (Mueller et al., 2000) and possibly the resulting immunostimulatory activity. Polysaccharides can have a large range of molecular weights (Greenwood, 1956). Their molecular weight distribution, structure, and size, all impact the behaviour of the polysaccharide. Gelpermeation chromatography (GPC), also known as size-exclusion chromatography (SEC), allows molecular weight determination based on hydrodynamic volume of polysaccharides (Gaborieau & Castignolles, 2011). The molecular weight of the sample can also be determined by dispersion by DOSY NMR (Suárez et al., 2006), or laser light scattering (Wang et al., 2009a), for example. Most of the studies stated the molecular weight as an important structural feature in a perspective of a source of structure–function relationships (Fig. 2b). The presence of functional groups, like acetyl and/or sulfate groups can affect the polysaccharides charge, solubility, and conformation (Mueller et al., 2000; Wang & Zhang, 2009). While acetyl groups promote the formation of hydrophobic pockets in polysaccharides, sulfates can provide negative charge. Depending of the patterns and types of substituents, different immunostimulatory responses can be triggered. The branching degree of the polysaccharides is a structural feature associated with the presence of linked monosaccharide residues or linked chains to their backbone. Immunostimulatory polysaccharides may have a linear backbone without branches or have more or less complex branches linked to their backbone. Depending on this structural feature, solubility and other structural features will be affected, namely molecular weight and conformation (Mueller et al., 2000).

4. Polysaccharides with reported immunostimulatory activity 4.1. Glucans Glucans are d-glucopyranosyl (d-Glcp) based polysaccharides (homoglucans) which, depending on their monosaccharide residues anomeric structure, can be ␣-d-glucans, ␤-d-glucans, and mixed ␣,␤-d-glucans (Synytsya & Novák, 2013) (Fig. 3). They also present different types of glycosidic bonds originating linear or branched either (␤1 → 4)-, (␤1 → 3)-, and (␤1 → 6)-d-glucans (Deng et al., 2013; Falch, Espevik, Ryan, & Stokke, 2000; Guo et al., 2009; Ohno, Miura, Chiba, Adachi, & Yadomae, 1995; Samuelsen et al., 2011; Satitmanwiwat et al., 2012; Smiderle et al., 2013; Wang, Zhang, Yu, & Cheung, 2009b; Zhang, Li, Xu, & Zeng, 2005; Zhao et al., 2010) or (␣1 → 3)-, (␣1 → 4)-, and (␣1 → 6)-d-glucans (Bao, Duan, Fang, & Fang, 2001; Luo et al., 2008; Nair et al., 2004; Sun et al., 2012; Yan, Wang, Li, & Wu, 2011; Zhao, Li, Luo, & Wu, 2006; Zhao, Kan, Li, & Chen, 2005). The complexity of glucans can further increase when there are monosaccharides present other than glucose (heteroglucans) (Fig. 3j–l), or structural differences in chain conformation, branching degree, molecular weight or presence of functional groups (Huang et al., 2012; Luo, Sun, Wu, & Yang, 2012; Qiao et al., 2009, 2010a; Wagner, Stuppner, Schäfer, & Zenk, 1988; Xu et al., 2012; Yi et al., 2012; Zha, Luo, Luo, & Jiang, 2007; Zhao et al., 2005). All these differences result in glucans with different structural

PS features (Mw, DS, conformation)

Animal/cell type

Immunostimulatory activity

LPS*

References

(␣1 → 6)-d-glucans branched at C-3 with Glcp (␤1 → 6)-d-glucans

Mw 14 kDa

BALB/c mice splenocytes

–

Zhao et al. (2006)

Mw 29 and 45 kDa

THP-1 cells

y

(␣1 → 6)-d-glucan

Mw 49.5 kDa

BALB/c peritoneal MO

y

Smiderle et al. (2013) Luo et al. (2008)

Chemically synthetized

Tetra- and pentaOligo-(␤1 → 3)-d-glucan

Mw 0.67–0.83 kDa; Not helical structures

BALB/c mice

–

Jamois et al. (2005)

Chemically synthetized

Oligo-(␤1 → 3)-d-glucanmannose

Mw 0.83–0.99 kDa; Not helical structures

BALB/c mice

–

Descroix et al. (2010)

Cordyceps sinensis (strain Cs-HK1)

Two (␣1 → 4)-d-Glcp: WIPS—branched with (␣1 → 6)-d-Glcp (∼14%) AIPS—linear glucan ((␣1 → 3)-d-Galp) (␣1 → 6)-d-mannoglucans (1 → 6)-branched, (␤1 → 3)-d-glucan Fucogalactoxyloglucan

MwWIPS 1180 kDa MwAIPS 1150 kDa Random coil structure

C57BL/6 mice inoculated with B16 cells

–

Yan et al. (2011)

Mw 22 kDa ␣-Glc 20% acetylated Mw 480 kDa Triple-helix Mw 25 kDa

BALB/c mice splenocytes and peritoneal MO Kunming (KM) mice inoculated with S180 cells C57BL/10 mice

↑ mitogenic and comitogenic activity ↑ splenocyte antibody production ↑ expression of pro-inflammatory genes ↑ NO, TNF-␣, IL-1b and IL-6 ↑ iNOS, TNF-␣, IL-1b and IL-6 mRNA ↑ phagocytosis activity of peritoneal MO ↑ influx of monocytes and granulocytes into the blood ↑ influx MO into the peritoneal cavity ↑% of granulocytes in peripheral blood, intra-peritoneal ↓ % of lymphocytes, intra-peritoneal ↑ influx of peritoneal MO ↑ phagocytic activity of peritoneal MO ↑ IL-2 by spleen cells ↑ antitumour ↑ immunostimulatory effects in splenocytes AIPS > > WIPS ↑IFN-␥ by splenocytes ↑TNF-␣ by MO ↑ Thymus and spleen indexes ↑ serum IL-2, IL-6, and TNF-␣ ↑ MO phagocytosis in vitro and in vivo

–

Zha et al. (2007)

–

Deng et al. (2013)

(␤1 → 3)-d-glucans (highly branched)

Mw 8 kDa

↑ MAPKs- and Syk-dependent TNF-␣ and IL-6 Dectin-1 recognition ↑ comitogenic activity ↑ anti-tumour activity

y

Sulfated (␤1 → 3)-d-glucan Carboxymethylated (␤1 → 3)-d-glucan

Mw 125 kDa; DSsulfate 0.94; stiff chain Mw 52 kDa; DScarboxymethyl 1.18 Mw 886–1090 kDa

CHO cells RAW264.7 cells; murine peritoneal MO; C57BL/6 and BALB/c nu/nu inoculated with Lewis lung cancer; BALB/c mice Splenocytes BALB/c mice inoculated with S-180 solid tumours

Wagner et al. (1988) Guo et al. (2009)

↑ thymus and spleen index

–

Wang et al. (2009b)

Human complement proteins

Activate complement system

y

KM mice

↑ splenocyte proliferation, ↑ acid phosphatase in peritoneal MO ↑ MO phagocytosis (HCPS-3  HCPS-1 and HCPS-2)

–

Samuelsen et al. (2011) Qiao et al. (2009, 2010a,b)

KM mice

↑ splenocyte proliferation ↑ NK cell cytotoxicity

–

Yi et al. (2012)

KM mice; YAC-1 cells

↑ proliferation of spleen cells ↑NK cell cytotoxicity ↑ phagocytic function of MO ↑ hemolytic activity ↑ serum IgG

–

Zhao et al. (2005)

Glucans Aconitum carmichaeli Agaricus bisporus and Agaricus brasiliensis Armillariella tabescens

Dendrobium huoshanense Dictyophora indusiata Echinacea purpurea Ganoderma lucidum

Hordeum vulgare Hyriopsis cumingii

(␤1 → 4)(␤1 → 3)-dglucans Arabinoglucan (HSCP-1) Glucan (HCPS-2) Galactorhamnoglucan with fucose (HCPS-3)

Imocarpus longan

Arabinomannoglucans LPI1 and LPI2

Ipomoea batatas (roots)

(␣1 → 6)-d-glucan

MwHCPS-1 432.2 kDa DSsulfate 0.3804% MwHCPS-2 457.9 kDa DSsulfate 0.5959% MwHCPS-3 503.1 kDa DSsulfate 6.2938% Not triple-helices Mw 14 kDa LPI1—sphere-like LPI2—single-helix Mw 53.2 kDa Compact random coil

S.S. Ferreira et al. / Carbohydrate Polymers 132 (2015) 378–396

PS name/structure

Source

382

Table 1 Different sources and structural features of immunostimulatory polysaccharides: glucans, mannans, pectic polysaccharides and arabinogalactan proteins, fucoidans, galactans, hyaluronans, fructans, and xylans.

RAW 264.7 cells

↑ NO, TNF-␣, and IL-6 by TLR2

–

Xu et al. (2012)

BALB/c mice inoculated with S-180 cells J774A.1 cells

↑ antitumour activity

–

Zhang et al. (2005)

↑ NO and TNF-␣

y

Luo et al. (2012)

Mw 17 kDa

ICR mice splenocytes

y

Sun et al. (2012)

(1 → 6)-branched, (␤1 → 3)-d-glucan (␤1 → 6)-d-Glc branched, (␤1 → 4)-d-Glc

Triple-helix

J774A.1 cells

↑ lymphocyte proliferation with or without LPS ↑ NO production ↑ NO and TNF-␣

y

Mw 35 kDa DSacetyl 0.1

KM mices inoculated with S18, hepatoma 22, and Ehrlich ascites carcinoma

Satitmanwiwat et al. (2012) Zhao et al. (2010)

(␤1 → 3)-d-glucan substituted with single (␤1 → 6)-d-Glcp residues at every third residue (1 → 6)-branched, (␣1 → 4)-d-glucan

Mw < 500 kDa or Mw> 1100 kDa Triple helix

Human monocytes

y

Falch et al. (2000)

Mw >550 kDa

Human lymphocytes; Human complement Kits

y

Nair et al. (2004)

Carboxymethylated (␣1 → 3)-d-glucan

Mw 80.4 kDa; DScarboxymethyl 0.28

Inbred ICR mice

Activate NK cells, T and B cells Complement activation Th1 pathway-associated profile ↑ lymphocyte proliferation; ↑ antibody production

–

Bao et al. (2001)

(␤1 → 4)-d-mannans

Mw 10,000 kDa, 1300 kDa, and 470 kDa DSacetyl 0.91

BALB/c mice

y

Leung et al. (2004)

(␤1 → 4)-d-mannans (G2E1DS3, G2E1DS2 and G2E1DS)

MwG2E1DS3 ≥ 400 kDa; 5 kDa ≤ MwG2E1DS2 ≤ 400 kDa; MwG2E1DS1 ≤ 5 kDa Mw140–90 kDa DSacetyl 0.08

RAW 264.7 cells

↑ peritoneal MO ↑ splenic T and B cell proliferation ↑ TNF-␣, IL-1␤, INF-␥, IL-2, and IL-6 (↑↑ for Mw 10,000 kDa) ↑ NO, TNF-␣, IL-1␤ by MO (G1E2DS1 and G1E2DS3 < G1E2DS2)

y

Im et al. (2005), Qiu et al. (2000)

C57BL/6 mice

↑ B lymphocyte activation

y

Simões et al. (2009, 2010)

Mw 109 DSacetyl 0.84

C57BL/6 mice

↑ B lymphocyte activation

y

Simões et al. (2009, 2010)

Mw 36 kDa Random coil

RAW 264.7 cells

↑ NO, IL-1␤, TNF-␣

y

Lee et al. (2010)

Mw 135 kDa DSsulfate 1.08% Mw 46 kDa Triple helix

RAW 264.7 cells

↑ TNF-␣ ↑ expression of COX-2 and iNOS ↑ NO, IL-1␤ and TNF-␣

–

Park et al. (2011)

y

Lee et al. (2009)

Mw 53 kDa

Lewis rats

–

Omarsdottir et al. (2005, 2006)

Mw 1350 kDa DSacetyl 0.03 DSsulfate 0.05 DSacetylated 0.23 DSdesacteyl 0

C57BL/6 mice; lymphocytes RAW264.7 cells

↑ splenocytes proliferation ↑ IL-10 secretion ↑ TNF-␣ by MO ↑ proliferation of spleen lymphocytes (TAPA1-s  TAPA1) ↑ NO by MO (TAPA1-ac> TAPA1> TAPA1-deac)

–

Du et al. (2009, 2010, 2014)

Arabinogalactoglucan

Lepista sordida

(1 → 6)-branched, (␤1 → 3)-d-glucan (␣1 → 6)-d-glucans (heteroglucan) (␣1 → 6)-d-glucan

Panax ginseng C. A. Meyer

Pleurotus sajor-caju Rhizobium sp. N613

Sclerotium rolfsii

Tinospora cordifolia

Unknown Mannans Aloe Vera

Coffea (infusion)

Coffea (spent coffee grounds) Cordyceps militaris

Haematococcus lacustris Hericium erinaceus (liquid culture broth) Peltigera canina

Tremella aurantialba (fruit bodies)

(␤1 → 4)-d-mannans with branches of (1 → 6)-d-Galp units (␤1 → 4)-d-mannans with branches of (1 → 6)-d-Galp units (␤1 → 6)-d-mannans with branches of (1 → 4)-d-Galp units Galactomannan (␤1 → 2)-d-mannans with side chain of (␤1 → 3)-d-Manp units (␣1 → 6)-d-mannan With (␣1 → 2)-d-Manp and (␤1 → 4)-d-Galp Xylomannans: (TAPA1) TAPA1-s (sulfonated) TAPA1-ac (acetylated) TAPA1-deac (deacetylated)

RAW 264.7 cells

↑ spleen and thymus weight ↑ phagocytic function of MO ↑ lymphocyte proliferation ↑ serum antibody ↑ TNF-␣ in monocytes

S.S. Ferreira et al. / Carbohydrate Polymers 132 (2015) 378–396

Mw 26 kDa Not triple-helix Mw 1490 kDa Triple-helix Mw 40 kDa

Lentinus edodes

383

384

Table 1 (Continued ) PS name/structure

PS features (Mw, DS, conformation)

Animal/cell type

Immunostimulatory activity

LPS*

References

Trigonella foenum-graecum L. (Fenugreek)

(␤1 → 4)-d-mannans with branches of (1 → 6)-d-Galp units (␣1 → 6)-d-Galp branched, (␤1 → 3)-d-mannans

Acetyl groups not detected

Sprague dawley rat; HB4C5 cells

–

Ramesh et al. (2001, 2002)

Mw 37 kDa

KM mice splenocytes

↑ phagocytosis by MO ↑ proliferation of MO ↑ IgM secretion in HB4C5 cells ↑ mitogenic and comitogenic activity

y

Sun et al. (2008)

AG-II

Mw 1600 kDa

↑ Phagocytosis ↑ ROS and TNF-␣

y

Moretão et al. (2004)

Artemisia tripartita

AG-II

Xie et al. (2008)

–

Fang and Chen (2013)

Mw 77.4 kDa

Inbred ICR mice splenocytes

↑ lymphocyte proliferation

–

Wang et al. (2005)

Chlorella pyrenoidosa

Branched rhamnogalacturonan type I (HAM-3-IIb-II) Rhamnogalacturonan (after deacetylation and carboxyl-reduction) AG

↑ ROS, NO, IL-6, IL-10, TNF-␣ and chemotactic protein-1 ↑ LPS-induced effect on B lymphocyte proliferation

y

Avicennia marina

Mw 251–49 kDa N- and O-acetylated DSacetyl 3.1%

Albino Swiss mice MO; S-180 cells; albino Swiss mices inoculated with S-180 cells J774.A1 cells; human and murine neutrophils Mice splenocytes

RAW264.7 cells

↑ NO

–

Coffea (instant coffee)

AGP

Mw 188 and 1020 kDa Not a rigid conformation Mw 5–6 kDa

Adult male guinea pigs (strain Trik); Balb/c mice

–

Cordyceps militaris

AG-I

Mw 576 kDa

BALB/c mice inoculated with Influenza A virus (NWS strain, H1N1); RAW 264.7 cells

Entada africana

AG-II

Mw 19 kDa

Sheep erythrocytes

Echinacea purpurea

Type II Acidic arabinogalactan

Mw 75 kDa

C57BL/10 mice, C3Hnu/nu mice

Antitussive ↑ TNF-␣, IL-2 and IFN-␥ by splenocytes ↑ survival rate of Influenza A virus infected mice ↑ TNF-␣ and IFN-␥ in treated mice ↑ NO by iNOS in MO ↑ mRNA expression of IL-1␤, IL-6, IL-10, and TNF-␣ by MO ↑ complement fixation activity (↓ after removal of T-Araf) ↑ IL-1, TNF-␣, and IFN-␤ by MO ↑ T cells proliferation

Suárez et al. (2006), Suárez et al. (2005) Nosál’ová et al. (2011)

Euterpe olerácea (fruit)

AG-II

Mw 4–800 kDa Presence of N- and O-acetyl groups

C57BL/6 or BALB/c mice

y

Glinus oppositifolius

AG-I and AG-II

Mw 70 kDa DSacetyl 4.3%

Sheep erythrocytes; PVG.7B strain rats lymphocytes; RNK-16 and mice MO; C3H/HeJ mice

Juniperus scopolorum

AG

J774.A1 cells

Lycium barbarian

AG-I

Mw 200–680 kDa N- and O-acetylated Mw 214.8 kDa

↑ IFN-␥ by NK and ␥␦ T cells in the lungs of C57BL/6 mice ↓ pulmonary Francisella tularensis and Burkholderia pseudomallei infections ↑ complement fixation activity B-lymphocytes proliferation ↑ IL-1␤ by MO ↑ mRNA for IFN-␥ in NK-cells ↑ proliferation of bone marrow cells through Peyer’s patch cells ↑ iNOS, NO, ROS, IL-1, IL-6, IL-12, TNF-a and IL-10 ↑ IgG by B-lymphocyte ↑ NF-␬B and AP-1expression B-lymphocytes proliferation

Polyporus albicans (Imaz.) Teng Pectic polysaccharides and arabinogalactan proteins Anadenanthera colubrina

Centella asiatica

Splenocytes

y

Ohta et al. (2007)

–

Diallo et al. (2001) Luettig et al. (1989), Wagner et al. (1988) Holderness et al. (2011), Skyberg et al. (2012)

y

Inngjerdingen et al. (2005, 2007a,b)

y

Schepetkin et al. (2005) Peng et al. (2001)

–

S.S. Ferreira et al. / Carbohydrate Polymers 132 (2015) 378–396

Source

–

Mao et al. (2005)

–

Togola et al. (2008)

y

PBMC

↑ proliferation of PBMC ↑ IL-1␤, TNF-␣, IL-10, IL-10, GM-CSF

–

Dourado et al. (2004) Yin et al. (2012)

J774.A1 cells; THP1-Blue cells; sheep erythrocytes; Human neutrophils

↑ ROS and NO by MO/monocytes ↑ TNF-␣ by MO ↑ NF-␬B in monocytes. ↑ complement-fixing activity stimulated MPO neutrophil release ↓ complement fixation activity ↑ complement fixation activity ↑ T cell independent induction of B-cell proliferation

y

Xie et al. (2007)

y

Nergard et al. (2005)

↑ IL-1␤, IL-6, IL-12, TNF-␣ by DCs and MO (Native  hypoS > deAc  hypoSdeAc)

y

Khil’chenko et al. (2011)

↑ NK cells activity ↑ lymphocyte proliferation

–

Zhou et al. (2004)

↑ NO

–

Suárez et al. (2010)

↑MO-phosphatase activity ↑TNF-␣, IL-6 ↑lysosomal activity of MO ↑ROS (␭-carregannan) ↑ weight of the thymus, spleen and lymphoid organ cellularity ↑ phagocytic activity ↑ neutrophil adhesion ↑ alkaline phosphatase activity ↓ Cy-induced myellosuppression ↑ phagocytosis, ↑ cytotoxicity by NK-cells, and antibody-dependent cell cytotoxicity ↑ lymphocyte proliferation.

–

Yermak et al. (2012)

–

Bhatia et al. (2013)

–

Stephanie et al. (2010)

–

Ke et al. (2013b)

–

Ke et al. (2013a)

Rhamnan

DSsulfate 21.8%

BALB/c mice

Opilia celtidifolia

Arabinogalacturonan

Mw 1000–8400 kDa

Prunus dulcis (seeds)

Arabinan-rich

Mw 762 kDa

Sheep erythrocytes; rat Wistar MO C57BL/6 mice spleen cells

Radix Astragali

Arabinan with AG-I and AG-II

Tanacetum vulgare

Acidic PS with AG-II

Mw 1334 kDa With O-acetyl groups Random coil Presence of N/O-acetyl groups

Vernonia kotschyana

Pectic polysaccharide (Vk100A2b) Pectic arabinogalactan (Vk100A2a)

Mw 1150 kDa DSacetyl 7% Mw 20 kDa DSacetyl 11%

Sheep erythrocytes; C3H/HeJ mice splenocytes

Fucoidan (native); Hyposulfated (hypoS); Deacetylated (deAc); Hyposulfated and deacetylated (hypoSdeAc)

Mw 150 kDa and 500 kDa

Balb/c mice

Galactans Chondrus ocellatus

Galactan (␭-carrageenans)

Mw 9.3–650 kDa Sulfate 21.8–30.5%

Chlorella pyrenoidosa

(␤1 → 3)-d-galactans

Acetylated

Gigartinaceae and Tichocarpaceae

Galactans (␬-,␤-,␫-,␭-carrageenans)

Mw 200–500 kDa Sulfate 20–28%

ICR mice inoculated with S180 and H22 cells; YAC-1 cells RAW 264.7 cells ICR mice human blood cells; BALB/C mice peritoneal fluid

Porphyra vietnamensis

Porphyran (sulfated galactan)

DSsulfate 1.15 DSmethyl 0.62

Wistar albino rats and albino mice; Sheep erythrocytes

Solieria chordalis

Galactans (carrageenans)

Mw< 20 kDa DSsulfate 33.54 ± 0.3

Daudi (Human Burkitt’s lymphoma); PBMC

Hyaluronans Streptococcus equi subsp. zooepidemicus

HA (CP-3)

Mw 1338.0 kDa

KM mice

Unknown

Hyaluronans

Mw 1050, 145, and 45.2 kDa

KM mice

Fucoidans Fucus evanescens

↑ splenocyte proliferation ↑ increase the activity of acid phosphatase in peritoneal MO ↑ splenocyte proliferation ↑ indices of spleen and thymus ↑ activity of lysozyme in serum (Mw145 and 45.2 > Mw1050 )

S.S. Ferreira et al. / Carbohydrate Polymers 132 (2015) 378–396

↑ spleen index, NK cytostatic activity and splenocytes activity ↑ complement fixing activity ↑ NO by MO ↑ lymphocyte activation markers

Monostroma angicava

385

386

Table 1 (Continued ) PS name/structure

PS features (Mw, DS, conformation)

Animal/cell type

Immunostimulatory activity

LPS*

References

Fructans Allium sativum (Aged extract)

Two fructans (HF and LF)

MwHF >3.5 kDa; MwLF <3 kDa

Chandrashekar et al. (2011)

Mw 1.1–1.2 kDa

↑ mitogenic activity ↑ intra-peritoneal MO activity ↑ phagocytosis of MO ↑ NK cell activity

–

(␤2 → 1)-d-fructooligosaccharides (␤2 → 1)-d-Fru branched, (␤2 → 6)-d-fructan

BALB/c mice and CFT Wistar rats PBMC

–

–

J744.1 RAW264.7 C3H/HeN and C3H/HeJ

↑ IL-12 and TNF-a

y

Borthakur et al. (2012) Xu et al. (2006)

(␤2 → 1)-d-fructans

–

BDF1 mice

y

Han et al. (2001)

Fructans

Mw 14 kDa Globular to helical fibrous shape at increasing concentrations

Balb/c mice

↑ IgM ↑ B cells proliferation ↑ iNOS mRNA and NO in MO ↑ lymphocytes proliferation

–

Wu et al. (2006)

4-O-methylglucuronoarabinoxylan

Mw 35 kDa

C57BL/10 mice, C3Hnu/nu mice

↑ IL-1 and oxygen radicals by MO ↑ B cells proliferation

–

Glucuronoxylans Aarabinoxylans Arabinoxylans (AXa and AXe)

Mw 21.5–990 kDa

Wistar rats thymocytes

↑ mitogenic and comitogenic activity

y

MwAXa 351,7 kDa MwAXe 32,52 kDa AXe had ferulic acid

BALB/c mice

↑ MO phagocytosis ↑ lymphocyte proliferation ↑ hypersensitivity reaction

y

Proksch and Wagner (1987), Stimpel et al. (1984) Ebringerová et al. (2002) Zhou et al. (2010)

Asparagus racemosus Linn. Bacillus subtilis (fermentation of soybeans) Platycodon grandiflorum

Ophiopogon japonicus

Xylans Echinacea purpurea

Several sources Triticum spp. (bran)

* LPS contamination evaluation or decontamination: y, evaluated; –, not evaluated. Abbreviations: PS, polysaccharides; Mw, molecular weight; DS, degree of substitution; LPS, lipopolysaccharide; R, references; MO, macrophages; TNF-␣, tumour necrosis factor ␣; IL, interleukin; iNOS, inducible nitric oxide synthase; mRNA, messenger ribonucleic acid; IFN, interferon; KM, Kunming; MAPK, mitogen-activated protein kinase; NK, natural killer; IgG, immunoglobulin G; TLR2, toll-like receptor 2; COX-2, cyclooxygenase-2; TP, total phenolics content; IgM, immunoglobulin M; PBMC, human peripheral blood mononuclear cells; GM-CSF, granulocyte-macrophage colonystimulating factor; AG-I, type I arabinogalactan; AG-II, type II arabinogalactan; AG, arabinogalactan; AGP, Arabinogalactan-protein: ROS, reactive oxygen species; NF-␬B, nuclear factor-␬B; AP-1, activator protein-1; MPO, myeloperoxidase; HA, hyaluronic acid; DCs, dendritic cells; Cy, cyclophosphamide.

S.S. Ferreira et al. / Carbohydrate Polymers 132 (2015) 378–396

Source

S.S. Ferreira et al. / Carbohydrate Polymers 132 (2015) 378–396

387

Fig. 2. (a) Number of papers from Scopus Database search with the topics “polysaccharides AND (immuno OR immune OR immunostimulatory OR immunomodulatory) AND type of polysaccharide” (white bars), and papers from these ones concerning also the topics of the following structural characteristics: “molecular weight”, functional groups (“sulfate OR acetyl OR Charge or charges”), branching degree (“ramification OR branch OR branches”), and/or “conformation” (black bars). (b) Percentage of papers associated to a structural characteristic for each type of polysaccharide given in (a). These searches cover papers from 1936 until 2014.

properties and therefore different interactions with the immune system (Vannucci et al., 2013). 4.1.1. Conformation Studies indicated that triple-helix conformation conferred higher immunostimulatory activity to ␤-d-glucans by promoting TNF-␣ release by monocyte or macrophage cells (Falch et al., 2000; Satitmanwiwat et al., 2012). The importance of triple-helix was also demonstrated when, after denaturation and renaturation, (␤1 → 3)-d-glucans with side chains of (␤1 → 6)-d-Glc with a less tight triple-helix conformation still enhanced cytokine production by human monocytes cultured in vitro (Falch et al., 2000) and in tumour-bearing mice (Deng et al., 2013). Although antitumour activities of polysaccharides may not be directly related with

immunomodulatory effects, the relevance of triple-helix in these polysaccharides was demonstrated (Wang et al., 2009b; Wang & Zhang, 2009), namely by the reduction of activity on murine sarcoma tumour growth inhibition by the destruction of the triple helix (Zhang et al., 2005). It was shown that also single-helix glucans had immunostimulatory activity as assessed by TNF-␣ increase in mouse serum, suggesting that the immunostimulatory activity of (␤l → 3)-dglucans may be depend on the existence of a helical conformation (Ohno et al., 1995). In contrast, heteroglucans without helical conformations also displayed stimulatory activity on immune cells (Huang et al., 2012; Xu et al., 2012). This suggests that the presence of other monosaccharides, namely arabinofuranosyl (Araf), galactopyranosyl (Galp),

Fig. 3. Illustration of chemical structure of several homoglucans: (a) cellulose, (b) linear (␤1 → 3)-d-glucans, (c) mixed ␤-d-glucans from cereals, (d) (␤1 → 3)-d-glucans branched at C-6 with Glcp (lentinan, scleroglucan, schizophylan, laminarinan) (e) zymosan, (f) bacterial glucan, (g) amylose, (h) dextran, (i) (␣1 → 4)-d-glucans branched at C-6 with (␣1 → 4)-d-Glcp chains (amylopectin, glycogen), and (j)–(l) heteroglucans (according to the nomenclature of carbohydrates proposed by McNaught (1997)).

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and mannopyranosyl (Manp) residues surpasses the requirement of helical conformations for the exhibition of immunostimulatory activity. 4.1.2. Molecular weight A diversity of studies on (␤1 → 3)-d-glucans with side chains of (␤1 → 6)-d-Glc report different ranges of molecular weight with immunostimulatory activities (Table 1), ranging from 8 kDa (Guo et al., 2009) to more than 1100 kDa (Falch et al., 2000). Moreover, it was suggested, by the study of (␤1 → 3)-d-Glcp oligosaccharides, that high molecular weight was not necessary to obtain immunostimulating effects (Descroix et al., 2010; Jamois et al., 2005). Such results suggest that the molecular weight is not an exclusive property, but is intrinsically related to other structural features, e.g. conformation of (1 → 3)-␤-d-Glcp rich structures (Mueller et al., 2000). 4.1.3. Functional groups Most glucans isolated from natural sources do not have functional groups, but there are a few exceptions. From these, (␤1 → 4)-d-glucans with side chains of C-3 acetylated (␤1 → 6)-dGlc units (Fig. 3f) (Zhao et al., 2010), acetylated (␣1 → 6)-d-glucans with Gal and/or Man residues (Fig. 3l) (Zha et al., 2007), and sulfated (␣1 → 4)-d-glucans with (␣1 → 4)-d-Glc branch chains attached to the backbone chain by 1 → 3 and 1 → 6 presented high immunostimulatory activity (Qiao et al., 2010b). These are contrasting with those (␣1 → 6)(␣1 → 4)-d-glucans (Fig. 3i) not acetylated nor sulfated, which do not present immunostimulatory activity (Maity et al., 2014). (␤1 → 3)-d-Glucans, when chemically functionalized to form their sulfated and carboxymethylated derivatives, improved solubility resulting in glucans with higher immunostimulatory activity (Bao et al., 2001). This effect may be explained by the impact of these groups in intramolecular and intermolecular hydrogen bonding, strengthening the effect of electrostatic repulsion and enabling the adoption of a certain structure. Furthermore, it was reported that glucans conformation was also modified after functionalization with sulfate and carboxymethyl groups, leading to stiffer chains (Bao et al., 2001; Wang et al., 2009b). It is important to note that this conformation, however, was less recognized by glucan receptors than the triple-helix conformation of non-sulfated (␤1 → 3)-d-glucans with side chains of (␤1 → 6)-d-Glc (Mueller et al., 2000). 4.1.4. Branching degree A high branching degree in ␤-d-glucans has been positively associated to immunostimulatory activity (Deng et al., 2013; Guo et al., 2009; Satitmanwiwat et al., 2012; Wang et al., 2009a). Highly branched (␤1 → 3)-d-glucans, with side chains of (␤1 → 6)-d-Glc, with an average of a side chain branch on every third glucose residue unit along the backbone, had higher immunostimulatory activity when comparing with less branched or linear (␤1 → 3)-dglucans (Deng et al., 2013; Satitmanwiwat et al., 2012; Wang et al., 2009a). However, it must be taken into account that to (␤1 → 3)-dglucans with more side chains of (␤1 → 6)-d-Glc were associated tighter triple-helix conformations, already described as important structural features for receptors recognition (Mueller et al., 2000). Therefore the effect of branching degree with (␤1 → 3)-d-glucans, which is a structural feature that can allow the formation of triple helices, seems to be a relevant characteristic for the promotion of the immunostimulatory activity of glucans. Moreover, both linear and/or branched non-like starch (␣1 → 6)-d-glucans and (␣1 → 4)-d-glucans have shown immunostimulatory activity (Luo et al., 2008; Nair et al., 2004; Sun et al., 2012; Zhao et al., 2005, 2006), suggesting that other structural characteristics may be involved, like conformation and molecular

weight. When comparing two (␣1 → 4)-d-glucans with similar conformations and molecular weight but different branches of short chains of (␣1 → 6)-d-Glcp, it was observed that the linear structure induced a higher in vitro lymphocyte proliferative effect (Yan et al., 2011), indicating that a lower branching degree was the structural feature responsible for the higher immunostimulatory activity. The immunostimulatory activity of heteroglucans is positively related with the branching degree. Either in ␣-d-heteroglucans or ␤-d-heteroglucans more branches of residues units or side chains of fucose, galactose, and/or mannose were associated to high immunostimulatory activity, in some studies confirmed by the loss of their activity after hydrolysis of their branches (Huang et al., 2012; Luo et al., 2012; Zhao et al., 2005). 4.1.5. Synopsis of glucans immunostimulatory activity (␤1 → 3)-d-Glucans may promote the immunostimulatory activity, as demonstrated in several cellular and in vivo assays. This activity was associated with the formation of triple helix conformations. Also, sulfation and carboxymethylation of (␤1 → 3)-d-glucans conferred immunostimulatory activity to polysaccharides, however not as high as that observed when the (␤1 → 3)-d-glucans form triple helices. Chains where linkages other than (␤1 → 3)-d-Glc, both glucans and heteroglucans are present may also show immunostimulatory activity, allowing inferring that the presence of short (␤1 → 3)-d-Glc structures may provide activity to different polysaccharides. Glucans not presenting (␤1 → 3)-d-Glc, if acetylated, may also display immunostimulatory activity. Several (␣1 → 6)(␣1 → 4)-d-glucans have also been reported to have immunostimulatory activity. However, in most of the studies, these compounds were not tested using in vivo models. This would be important as their structural features responsible for immunostimulatory activity could be modified upon digestion by ␣-d-amylases present in saliva and pancreatic juice (Volman, Mensink, van Griensven, & Plat, 2010). Moreover, it is not known what distinguishes these immunostimulatory (␣1 → 6)(␣1 → 4)-dglucans from non-active starch (Maity et al., 2014). 4.2. Mannans Immunostimulatory mannans are polysaccharides with a backbone of Manp residues that can be more or less ramified with other monosaccharides. The backbone of immunostimulatory mannans mainly consists of (␤1 → 4)-d-Manp (Im et al., 2005; Leung et al., 2004; Qiu, Jones, Wylie, Jia, & Orndorff, 2000; Ramesh, Yamaki, Ono, & Tsushida, 2001; Ramesh et al., 2002; Simões et al., 2009; Simões, Nunes, Domingues, & Coimbra, 2010, 2012) (Fig. 4a), (␤1 → 3)-dManp (Sun et al., 2008) (Fig. 4b) or (␣1 → 3)-d-Manp (Du et al., 2009, 2010, 2014) (Fig. 4c), (␤1 → 2)-d-Manp (Lee, Cho, & Hong, 2009) (Fig. 4d), and (␤1 → 6)-d-Manp (Lee et al., 2010) (Fig. 4e and f) or (␣1 → 6)-d-Manp (Omarsdottir, Freysdottir, Barsett, Smestad Paulsen, & Olafsdottir, 2005; Omarsdottir et al., 2006) (Fig. 4g). Structural differences can also arise from the degree and sequence in which these possible backbones are substituted by various side chains containing residues of ␣- and ␤-d-Galp, d-Manp, or dGlcp, and/or functional groups, like acetyl groups (Chlubnová et al., 2011). 4.2.1. Conformation Galactoglucomannans composed mainly by (␤1 → 6)-d-Manp, with random coil conformation, have high immunostimulatory activity (Lee et al., 2010). Nevertheless, a triple-helix conformation mannan composed by (␤1 → 2)-d-Manp branched with (␤1 → 3)d-Manp (Lee et al., 2009) (Fig. 4d), was described having less activity

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Fig. 4. Illustration of chemical structure of possible immunostimulatory mannans: (a) (␤1 → 4)-d-mannan (coffee galactomannans); (b) (␤1 → 3)-d-mannan; (c) (␣1 → 3)d-mannan; (d) (␤1 → 3)-branched, ␤-(1 → 2)-d-mannan; (e) and (f) (␤1 → 6)-d-mannans; and (g) (␣1 → 6)-d-mannan.

than the (␤1 → 6)-d-Manp random coil of the galactoglucomannan (Lee et al., 2010) (Fig. 4f). 4.2.2. Molecular weight Although the reports suggesting high immunostimulatory activity of (␤1 → 4)-d-mannans associated to higher molecular weight (10 MDa) acetylated polysaccharides when compared with those with 1.3 MDa with the same degree of acetylation (Leung et al., 2004; Pugh, Ross, ElSohly, & Pasco, 2001), the majority of (␤1 → 4)-d-mannans immunomodulatory activities studied refer to polysaccharides with molecular weight in the range between 5 and 400 kDa (Im et al., 2005; Qiu et al., 2000; Simões et al., 2009, 2010). This range of molecular weight is also reported for (␤1 → 3)-, (␣1 → 3)-, and (␣1 → 6)-d-mannans with immunostimulatory activity (Lee et al., 2009; Sun et al., 2008), showing that the differences in molecular weight could be not as relevant as other structural features. 4.2.3. Functional groups A relationship of acetyl groups and the immunostimulatory function has been identified for naturally occurring and chemically acetylated (␤1 → 4)-d-mannans (Leung et al., 2004; Qiu et al., 2000; Simões et al., 2009). The importance of acetyl groups was reinforced as non-acetylated (␤1 → 4)-d-mannans did not show

immunostimulatory activity. On the other hand, the position of the acetyl group seems not to be an essential feature in (␤1 → 4)-dmannans, since both, more acetylated in their backbone or in side chains, showed similar immunostimulatory activity (Simões et al., 2009, 2010; Simões et al., 2012). The importance of acetyl groups was also described for (␣1 → 3)-d-mannans (Du et al., 2014). The presence of sulfate groups was also described in immunostimulatory mannans (Park et al., 2011). Additionally, chemically sulfated mannans were markedly more stimulators than the original ones (Du et al., 2010). 4.2.4. Branching degree Similar branching degrees were found in structurally different immunostimulatory mannans, namely, a (␤1 → 3)-d-linked side chain mannan linked to a (␤1 → 2)-d-mannan backbone (Lee et al., 2009) (Fig. 4d) presented higher stimulatory activity than mannan with a backbone of (␤1 → 6)-d-Manp residues, with branches of (1 → 4)-d-Galp units (Lee et al., 2010) (Fig. 4f). It is thus possible that the kind of branching could be important for the immunostimulatory activity of mannans. However, knowing that other structural features like acetylation or sulfation could more significantly affect this activity than different branching degrees (Du et al., 2009, 2010, 2014), further studies are needed to determine how the branching degree could affect immune stimulation.

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Fig. 5. Illustration of chemical structure of the primary structure of pectic polysaccharides according to the models proposed by (a) Pérez et al. (2003) and (b) Vincken et al. (2003).

4.3. Pectic polysaccharides and arabinogalactan proteins Pectic polysaccharides are complex heteropolysaccharides, which have in common a high proportion of galactopyranosyluronic acid (GalpA), and can be found in plants (Visser & Voragen, 1996). Pectic polysaccharides include various fragments of linear and ramified regions covalently connected. From the several models proposed (Yapo, 2011), the most consistent are those suggested by Pérez, Rodríguez-Carvajal, and Doco (2003) and Vincken et al. (2003) (Fig. 5). The linear region consists of units of (␣1 → 4)-d-GalpA residues (HG, homogalacturonan region) that can carry methyl ester groups and can also be acetylated at the galacturonan backbone (Mao et al., 2005). A backbone of alternating (␣1 → 4)-d-GalpA and (␣1 → 2)-l-Rhamnopyranosyl (l-Rhap) residues, ramified in the Rha by galactans (Grønhaug et al., 2011), type I arabinogalactans (Ohta et al., 2007; Peng, Huang, Qi, Zhang, & Tian, 2001) and type II arabinogalactans (Grønhaug et al., 2011) (Fig. 6), arabinans (Cardoso, Ferreira, Mafra, Silva, & Coimbra, 2007; Dourado et al., 2004; Yin et al., 2012), of varying structure is named type I rhamnogalacturonans (RG-I). Also, structures containing single xylose (Xyl) or apiose (Api) residues as side chains at the galacturonan backbone have been called xylogalacturonans (XG) and apioglacturonans (ApG). Type II rhamnogalacturonans (RG-II) are branched structures composed of several monosaccharides, including 2-O-methylfucose, 2-O-methylxylose, and apiose, usually not observed in other polysaccharides (Diallo, Paulsen, Liljebäck, & Michaelsen, 2003; Fang & Chen, 2013; Vincken et al., 2003; Wang et al., 2005). Therefore, structural diversity arises from the branching degree, degree of methyl esterification, degree of acetylation, the type of branched chains and molecular weight (Popov & Ovodov, 2013). The review of Waldron and Faulds (2007)

contains detailed information on the structural features of each one of these individual polysaccharides, general properties, extraction conditions, and role in plant cell walls. Type I arabinogalactans (AG-I) are arabinosyl-substituted derivatives of linear (␤1 → 4)-d-Galp units. ␣-l-Araf and ␤-dGalp units can be linked via position 3 along the main chain (Ohta et al., 2007; Peng et al., 2001). AG-I are found as ramified regions of rhamnogalacturonan backbones in pectic polysaccharides (Inngjerdingen et al., 2007a; Peng et al., 2001). Type II arabinogalactans (AG-II) comprise highly branched polysaccharides with ramified chains of (1 → 3)-linked and (1 → 6)linked ␤-d-Galp units, the former predominantly in the interior and the latter in the exterior chains (Diallo et al., 2003; Holderness et al., 2011; Moretão, Zampronio, Gorin, Iacomini, & Oliveira, 2004; Luettig, Steinmuller, Gifford, Wagner, & Lohmann-Matthes, 1989; Nergard et al., 2005; Skyberg et al., 2012; Xie, Schepetkin, & Quinn, 2007; Xie et al., 2008) (Fig. 6b). The arabinosyl units might be attached through different positions of the (␤1 → 6)-d-Galp side chains. AG-II may occur as side chains of pectic polysaccharides, or in a complex family of proteoglycans known as arabinogalactanproteins (AGP) (Diallo, Paulsen, Liljebäck, & Michaelsen, 2001; Nosál’ová et al., 2011; Nunes, Reis, Silva, Domingues, & Coimbra, 2008; Suárez et al., 2006). This kind of structures can be easily identified by the Yariv reagent assay (Holderness et al., 2011; Schepetkin et al., 2005, 2008; Togola et al., 2008; Xie et al., 2007). 4.3.1. Conformation Immunostimulatory pectic polysaccharides with arabinogalactan structures exhibited random coil conformations (Suárez et al., 2006; Yin et al., 2012). In this kind of polysaccharides their

Fig. 6. Illustration of chemical structure of (a) type I and (b) type II arabinogalactans.

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Fig. 7. Illustration of chemical structure of some galactans: (a) ␬-carrageenan, (b) ␭-carrageenan, (c) ␫-carrageenan, (d) ␤-carrageenan, and (e) porphyran.

activity was associated to a flexible chain conformation and not rigid conformations. 4.3.2. Functional groups Acetyl groups were identified in immunostimulatory pectic polysaccharides (Fang & Chen, 2013; Nergard et al., 2005; Wang et al., 2005). A higher degree of acetylation, due to acetyl groups localized in the galacturonan backbone, has been associated to lower immunostimulatory activity, shown by an increase of activity after a deacetylation procedure (Wang et al., 2005). This behaviour contrasted with the reported higher immunostimulatory activity associated to glucans and mannans previously mentioned. This may be explained by the immunosuppressive activity that has been reported for deesterified homogalacturonan backbones (Popov & Ovodov, 2013), where a high content of negatively charged carboxyl groups from galacturonic acid residues occur. Acetyl groups were identified in AG-II structures displaying macrophage stimulatory activity (Schepetkin et al., 2005; Xie et al., 2007, 2008) and in mixed type I and II structures with stimulatory effect on cytokine production by human peripheral blood mononuclear cells (Yin et al., 2012). Additionally, sulfated pectic polysaccharides also showed stimulatory activity on diverse macrophage and neutrophil effector functions, as shown by several sulfated AG- II, with at least 3.4% of sulfate groups (Xie et al., 2008). 4.3.3. Branching degree The isolated branched regions of pectic polysaccharides, characterized as galactans, arabinans (Dourado et al., 2004; Fang & Chen, 2013), and type I arabinogalactans, are the main responsible for the resulting immunostimulatory activity (Akhtar et al., 2012; Capek & Hribalova, 2004; Ebringerová, Hromádková, ´ Alfödi, & Hˇrıbalová, 1998; Ebringerová, Kardoˇsová, Hromádková, ´ ´ Malovíková, & Hˇríbalová, 2002; Kardoˇsová, Malovıková, Pätoprsty, Nosál’ová, & Matáková, 2002; Popov & Ovodov, 2013; Zhou et al., 2010). In fact, the removal of the linear regions by enzymatic treatments with endo-polygalacturonase resulted in higher immunostimulatory activity (Togola et al., 2008) whereas the treatment with exo-␣-l-arabinofuranosidase and exo-␤-dgalactosidase, with removal of the branching regions diminished the pectic polysaccharides immunostimulatory activity (Nergard et al., 2005). Also, the same behaviour was obtained by removal of Araf residues by weak acid hydrolysis (Diallo et al., 2001, 2003; Duan, Dong, Ding, & Fang, 2010; Inngjerdingen et al., 2007a; Wang et al., 2005). More information can be found in Yamada and Kiyohara (2007), where relationships of pectic polysaccharides

anticomplementary, immune complex clearance, mitogenic, and intestinal immune system-modulating activities were described in more detail. 4.4. Galactans Galactans are polysaccharides rich in galactose. Besides the type I and II arabinogalactans described in Section 4.3, other galactans, derived from marine organisms, such as the carrageenans (Fig. 7a–d) (Stephanie, Eric, Sophie, Christian, & Yu, 2010; Yermak et al., 2012; Zhou et al., 2004) and porphyrans (Fig. 7e) (Bhatia et al., 2013), have also been studied concerning their immunostimulatory activity. Carrageenans are chemically characterized by repeating disaccharide units, consisting of sulfated or unsulfated d-galactose residues that are linked in alternating (␤1 → 4)- and (˛1 → 3)bonds. There are several carrageenans, classified according to the presence of the 3,6-anhydro-bridge on the 4-linked galactose residue, and position and number of sulfate groups (Stephanie et al., 2010; Yermak et al., 2012; Zhou et al., 2004). Porphyrans are characterized by a linear backbone consisting of 3-linked ␤-d-galactosyl units alternating with either 4-linked ␣-l-galactosyl 6-sulfate or 3,6-anhydro-␣-l-galactosyl units (Bhatia et al., 2013). 4.4.1. Molecular weight Low molecular weight (<20 kDa) fractions of carrageenans are associated to higher immunostimulating properties (Stephanie et al., 2010; Zhou et al., 2004). Moreover, the low molecular weight is also associated to lower viscosity, which facilitates the immunostimulatory assays (Zhou et al., 2004). 4.4.2. Functional groups The immunostimulating activity of galactans from algae, affecting cellular and humoral immune response parameters, has been attributed to its high degree of sulfation (Bhatia et al., 2013; Stephanie et al., 2010; Yermak et al., 2012; Zhou et al., 2004). With such a high impact on immune system activation, they can even trigger, after long term exposition, uncontrolled pro-inflammatory responses with associated harmful effects (Bhattacharyya et al., 2013; Borthakur et al., 2012). The studies on the influence of acetyl groups in galactans and their influence on the immunostimulatory activity are scarce, although they have also been associated with algae mannogalactans stimulatory effect on NO production by macrophage cells activity (Suárez, Kralovec, & Grindley, 2010).

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Fig. 8. Illustration of the chemical structure of: (a) fucoidan, (b) hyaluronan, (c) fructans of (c1) inulin, (c2) levan, and (c3) mixed type, and (d) xylans, which include the (d1) glucuronoxylans and (d2) arabinoxylans.

4.5. Fucoidans Fucoidans refers to sulfated fucans, that is, sulfated rich lFucopyranosyl (l-Fucp) polysaccharides (Li, Lu, Wei, & Zhao, 2008). It is generally recognized that fucoidans are heteropolysaccharides made of l-Fucp (35–50%), (␣1 → 2)-, (␣1 → 3)- or (␣1 → 4)-linked, that can be sulfated or acetylated at various positions (Fig. 8a). The other monosaccharides that can be present are Galp, Manp, Xylp and uronic acids (Chlubnová et al., 2011). The naturally higher content of sulfate groups in fucoidans is associated with a higher stimulatory activity on macrophage cells (Qiao et al., 2010a,b; Teruya, Takeda, Tamaki, & Tako, 2010). Moreover, removal of almost every sulfate groups lead to a markedly reduced activity (Khil’chenko et al., 2011). The presence of acetyl groups also seems to be an important structural feature, because, after a deacetylation treatment, a decrease in the stimulatory activity of fucoidans on cytokine production by bone-marrow-derived dendritic cells and macrophages was observed (Khil’chenko et al., 2011). Furthermore, the simultaneous presence of acetyl and sulfate groups was crucial for fucoidans activity, since a prepared deacetylated and hyposulfated fucoidan lost almost all activity (Khil’chenko et al., 2011).

the resulting hyaluronans with 45.2 and 145 kDa exhibited much stronger immunostimulatory activity (Ke et al., 2013a). 4.7. Fructans Fructans are reserve carbohydrates comprising up to 70 units of fructose, linked to a terminal sucrose molecule. According to the type of linkage, fructans are classified into three families (Benkeblia, 2013), namely, inulin [(␤2 → 1)-d-Fruf] (Fig. 8c1), levan [(␤2 → 6)-d-Fruf] (Fig. 8c2), and mixed type [both (␤2 → 1)- and (␤2 → 6)-linked d-Fruf] (Fig. 8c3). Oligosaccharides of the fructans type act as bifidogenic agents and immune system stimulators associated with the intestinal mucosa (Delgado, Tamashiro, & Pastore, 2010). 4.7.1. Conformation An association of helical conformation with lymphocyte stimulatory activity was showed for fructans. This conformation was evidenced at increasing mixed-type fructan concentration by atomic force microscopy, showing a concentration-dependent formation of helical conformation which associated with a higher lymphocyte proliferative activity in vitro (Wu et al., 2006).

4.6. Hyaluronans Hyaluronan, also known as hyaluronic acid, is a major carbohydrate component of the extracellular matrix of mammalian tissue and can be found in skin, joints, eyes, and many other organs and tissues (Tzianabos, 2000), but can also be found in other sources. It contains a disaccharide repeating unit of N-acetylglucosamine (GlcNAc) and GlcA (Fig. 8b) and has been associated to immunostimulatory activity (Ke et al., 2013a,b). Hyaluronans with molecular weight of 1050 and 1338 kDa showed stimulatory activity on diverse immune cells (Ke et al., 2013b). However, a size-effect study showed that after hydrolysis,

4.7.2. Molecular weight According to fructans definition, these kind of polysaccharides have up to 70 units and, considering that each unit contributes with 0.180 Da to the polysaccharide molecular weight, they have up to 13 kDa. Therefore, only molecular weights under 13 kDa have been studied (Borthakur et al., 2012; Chandrashekar, Prashanth, & Venkatesh, 2011; Han et al., 2001; Hosono et al., 2003; Wu et al., 2006; Xu et al., 2006). The absence of studies with fructans with higher molecular weights does not allow concluding about the influence of molecular weight on fructans’ immunostimulatory activity.

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4.8. Xylans Xylans are polysaccharides present in plant cell walls and contain predominantly a backbone of (␤1 → 4)-d-Xylp residues units linked. These polysaccharides contain other sugar monomers attached to their backbone, including ␣-d-GlcpA units (glucuronoxylans, Fig. 8d1) and ␣-l-Araf units (arabinoxylans, Fig. 8d2). Several authors reported immune stimulatory activity for these polysaccharides either in vivo or in vitro, although not relating their structures with the stimulatory properties (Akhtar et al., 2012; Ebringerová et al., 1998, 2002; Kardoˇsová et al., 2002; Stimpel, Proksch, Wagner, & Lohmann-Matthes, 1984; Zhou et al., 2010). 5. Concluding remarks Immunostimulatory activities of polysaccharides may be due to direct or indirect interactions with immune system components, triggering diverse cellular and molecular events. Complement proteins and monocytes, macrophages, dendritic cells, neutrophils and lymphocytes have been reported as targets responding to polysaccharides. A direct stimulatory effect on these immune cells involves specific recognition receptors that can ultimately determine the resulting response. These receptors can be found on the cell membrane of phagocytic cells, as shown for macrophages and dendritic cells, and include scavenger receptors (SRs), Dectin-1, mannosereceptor, CR3, and TLR4. Upon binding of polysaccharides to these receptors, diverse signalling pathways may be triggered, leading to detectable responses. These have majorly been shown using in vitro assays but polysaccharide effects have also been described in vivo. In vitro assays evaluating the interaction of polysaccharides with the complement system have been the main non cellular methods used to evaluate immunological activity of polysaccharides, in particular, pectic polysaccharides. In these assays, incubation of human serum with active polysaccharides resulted in a decreased hemolysis of antibody-sensitized sheep erythrocytes as well as in the formation of complement activation products (Michaelsen, Gilje, Samuelsen, Høgåsen, & Paulsen, 2000). Complement activator polysaccharides may thus be referred as having anticomplementary activity, as inhibition of erythrocyte lysis may follow reduced complement titer ensuing from the activation of the complement system (Yamada & Kiyohara, 2007). In addition, murine cell lines, such as the macrophage cell line Raw 264.7, or murine splenocytes provided most of the evidence supporting the immunostimulatory activities reported. Production of reactive oxygen or nitrogen species, cytokine production, and cellular proliferation are common readouts in assessing the immunostimulatory activity of polysaccharides that could be evaluated by colorimetric assays. Polysaccharides may also induce the expression of activation markers on immune cells surface or intracellularly, that can be easily measured by using fluorescencelabelled monoclonal antibodies and flow cytometry. Although in vitro studies can provide valuable information towards the identification of target cells, signalling pathways involved in the polysaccharides effects, and elicited effector functions, most of the reported observations remain phenomenological and are often overinterpreted. More complex and truly functional in vitro assays as well as in vivo studies will be necessary to more extensively and accurately assess the impact of in vitro-detected activities on immune function. The usage of in vitro systems using specific gene-deficient or transgenic cells or in vivo assays with mutant mouse strains could provide powerful mechanistic information on how polysaccharides may modulate the immune response and better evaluate their potential usage such as in functional foods. Nevertheless, in order to find structure–function relationships, the selective modification of structural features on immunostimulatory polysaccharides should be first considered

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before proceeding to in vivo studies. Despite stimulatory effects of polysaccharides on antigen-presenting cells have been increasingly reported, how these interactions could affect polarization of responding antigen-specific T cells and the course of antigenspecific immune responses are questions that have been only marginally addressed. Moreover, although immunostimulatory effects of polysaccharides have been widely reported, putative effects in down-regulating immune responses are yet to be explored. Recent studies showing profound effects of fungal ␤-glucans in epigenetic regulation of immunological pathways responsible for trained immunity show how deep polysaccharides can impact on immune function and highlight the complexity of analytical tools necessary to uncover and explore polysaccharides effects (Saeed et al., 2014; Cheng et al., 2014). Glucans, mannans, pectic polysaccharides, arabinogalactans, fucoidans, galactans, hyaluronans, fructans, and xylans are polysaccharides with reported immunostimulatory activity. A literature survey on the structural features of the polysaccharides and reported immunostimulatory activity is critically presented here. Several structural features have been identified as responsible for immunostimulatory activity of specific polysaccharides. The chemical structure (type of polysaccharide), but also molecular weight, conformation, functional groups (namely acetyl and sulfate groups), and branching were some of the structural features identified as determining or contributing to the polysaccharides immunostimulatory activity. Furthermore, not only the individual, but also the combinations of these structural features seem to influence their immunostimulatory activity. From these structural features, the presence of triple-helix conformation of (␤1 → 3)d-glucans, the presence of acetyl and sulfate groups of mannans, galactans, and fucoidans, the presence of branching degree of pectic polysaccharides, and the existence of Type II arabinogalactans are crucial structural features for their immunostimulatory activity. The information reviewed here may be helpful in the definition of structure–function relationships necessary to design immunostimulatory polysaccharides with potential for therapeutical use or to be used as ingredients in functional foods. In parallel with in vivo immunological studies, the assessment of physiological effects of polysaccharides consumption will also be necessary aiming at the development of functional ingredients, novel therapeutic agents or adjuvants for preventing or treating immune-related pathological conditions that are important health challenges worldwide. Acknowledgements Thanks are due to Fundac¸ão para a Ciência e a Tecnologia (FCT, Portugal), European Union, QREN, FEDER and COMPETE for funding the QOPNA research unit (project PEst-C/QUI/UI0062/2013; FCOMP-01-0124-FEDER-037296). Cláudia Passos was supported by a post-doc grant by FCT (SFRH/BDP/65718/2009). References Akhtar, M., Tariq, A. F., Awais, M. M., Iqbal, Z., Muhammad, F., Shahid, M., et al. (2012). Studies on wheat bran Arabinoxylan for its immunostimulatory and protective effects against avian coccidiosis. Carbohydrate Polymers, 90, 333–339. Bao, X., Duan, J., Fang, X., & Fang, J. (2001). Chemical modifications of the (1 → 3)␣-d-glucan from spores of Ganoderma lucidum and investigation of their physicochemical properties and immunological activity. Carbohydrate Research, 336, 127–140. Benkeblia, N. (2013). Fructooligosaccharides and fructans analysis in plants and food crops. Journal of Chromatography A, 1313, 54–61. Bhatia, S., Rathee, P., Sharma, K., Chaugule, B. B., Kar, N., & Bera, T. (2013). Immunomodulation effect of sulphated polysaccharide (porphyran) from Porphyra vietnamensis. International Journal of Biological Macromolecules, 57, 50–56. Bhattacharyya, S., Xue, L., Devkota, S., Chang, E., Morris, S., & Tobacman, J. K. (2013). Carrageenan-induced colonic inflammation is reduced in Bcl10 null mice and increased in IL-10-deficient mice. Mediators of Inflammation, 1–13. I.D. 397642. Borthakur, A., Bhattacharyya, S., Anbazhagan, A. N., Kumar, A., Dudeja, P. K., & Tobacman, J. K. (2012). Prolongation of carrageenan-induced inflammation in human

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