Toll-like receptor 4-related immunostimulatory polysaccharides: Primary structure, activity relationships, and possible interaction models

Accepted Manuscript Title: Toll-like receptor 4-related immunostimulatory polysaccharides: Primary structure, activity relationships, and possible interaction models Author: Xiaorui Zhang Chunhui Qi Yan Guo Wenxia Zhou Yongxiang Zhang PII: DOI: Reference:

S0144-8617(16)30474-X http://dx.doi.org/doi:10.1016/j.carbpol.2016.04.097 CARP 11037

To appear in: Received date: Revised date: Accepted date:

27-1-2016 18-4-2016 21-4-2016

Please cite this article as: Zhang, Xiaorui., Qi, Chunhui., Guo, Yan., Zhou, Wenxia., & Zhang, Yongxiang., Toll-like receptor 4-related immunostimulatory polysaccharides: Primary structure, activity relationships, and possible interaction models.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.04.097 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Toll-like receptor 4-related immunostimulatory polysaccharides: Primary structure, activity relationships, and possible interaction models

Xiaorui Zhang, Chunhui Qi, Yan Guo, Wenxia Zhou*, Yongxiang Zhang

Beijing Institute of Pharmacology and Toxicology, Beijing 100850, RP China Highlights



   

In this review, we first summarize the monosaccharide type of different origin polysaccharides related to TLR4, including high plant origin polysaccharides, fungus origin polysaccharides, bacterial origin polysaccharides, alga origin polysaccharides, and animal origin polysaccharides. Second, we briefly describe the glucosidic bond types of TLR4 related heteroglycans and homoglycans. Third, the polysaccharides molecular weight ranges are summarized. Fourth, the primary structures and activity relationships of polysaccharides with TLR4/MD-2 are also discussed. Last, based on the existing interaction models of LPS with TLR4/MD-2 and linear polysaccharides with proteins, we speculate the possible interaction models of polysaccharide ligands with TLR4/MD-2.

Contents 1. Introduction ...................................................................................................................... 6 2. Monosaccharide composition .......................................................................................... 8 2.1. Higher plant polysaccharides ................................................................................ 8 2.2. Fungal polysaccharides......................................................................................... 9 2.3. Bacterial polysaccharides.................................................................................... 10 2.4. Algal and animal polysaccharides ....................................................................... 11 3. Heteroglycan glucosidic bonds ...................................................................................... 17 3.1. Polymers composed of two monosaccharaide types.......................................... 17 3.1.1. Glucose and fucose polymers................................................................... 17 3.1.2. Polymers of glucose and fructose ............................................................. 18 3.1.3. Glucose and mannose polymers .............................................................. 18 3.1.4. Galactose and rhamnose polymers .......................................................... 20 3.2. Polymers containing three types of monosaccharides ....................................... 21 3.2.1. Glucose, galactose, and mannose polymers............................................ 21 3.2.2. Glucose, glucose aldehyde acid, and mannose polymers ....................... 22 3.2.3. Glucose, glucose aldehyde acid, and fucose polymers ........................... 23 3.2.4. Glucose, galactose, and arabinose polymers........................................... 24 3.2.5. Glucose aldehyde acid, mannose, and xylose polymers ......................... 24 3.2.6. Glucose aldehyde acid, fucose, and xylose polymers.............................. 25 3.2.7. Galactose, galacturonic acid, and arabinose polymers ............................ 25 3.2.8. Galactose, arabinose, and rhamnose polymers ....................................... 26 3.3. Polymers with four monosaccharaide types ....................................................... 27 3.3.1. Galactose, arabinose, galacturonic acid, and rhamnose polymers.......... 27 3.4. Polymers with five monosaccharaide types ........................................................ 27 3.4.1. Galactose, galacturonic acid, rhamnose, arabinose, and xylose polymers ............................................................................................................................. 27 3.4.2. Glucose, galactose, mannose, rhamnose, and xylose polymers ............. 28 3.5. Polymers with six monosaccharaide types ......................................................... 29 3.5.1. Glucose, galactose, mannose, rhamnose, arabinose, and galacturonic acid

polymers .............................................................................................................. 29 3.5.2. Glucose, galactose, galacturonic acid, mannose, xylose, and fucose polymers .............................................................................................................. 29 3.5.3. Glucose, Glucuronic acid, galactose, Galacturonic acid, mannose, and xylose polymers................................................................................................... 30 3.5.4. Glucose, galactose, mannose, rhamnose, arabinose, and xylose polymers ............................................................................................................................. 30 4. Homoglycan glucosidic bonds ....................................................................................... 31 4.1. Glucan ................................................................................................................. 31 4.1.1. ß-(1,3)-D-Glucan polymers ....................................................................... 32 4.1.2. ß-(1,4)-D-Glucan polymers ....................................................................... 32 4.1.3. α-(1,4)-D-glucan polymers ........................................................................ 32 4.2. Galactan .............................................................................................................. 33 4.2.1. α-(1,3) and β-(1,4)-galactose polymers .................................................... 33 4.2.2. α-(1,4)-galacturonic acid polymers ........................................................... 34 4.3. Mannan ................................................................................................................ 34 4.4. Fructan................................................................................................................. 35 4.5. Polysialic acid ...................................................................................................... 36 5. Molecular weights .......................................................................................................... 39 6. Immune target cells ....................................................................................................... 42 7. Primary structure and activity relationships ................................................................... 45 7.1. Monosaccharide types and activity relationships................................................ 45 7.2. Glucosidic bonds and activity relationships ........................................................ 46 7.3. MW and activity relationships ............................................................................... 47 8. Polysaccharides and protein interaction models ........................................................... 49 9. Conclusions and outlook................................................................................................ 54 Acknowledgments ...................................................................................................... 55 References ................................................................................................................. 55

Nomenclature APC

Antigen-presenting cell

Ara

Arabinose

CD

Cluster of differentiation

CPS

Capsular polysaccharides

CR3

Complement receptors 3

DC

Dendritic cell

dp

Degree of polymerization

ERK

Extracellular signal-regulated kinase

Gal

Galactose

GalA

Galacturonic acid

GalNAc

N-acetylgalactosamine

Glc

Glucose

GlcNAc

N-acetylglucosamine

GluA

Glucuronic acid

HPLC

High performance liquid chromatography

ICAM-1

Intercellular adhesion molecule-1

IFN

Interferon

iNOS

Inducible nitric-oxide synthase

JNK

C-Jun NH2 terminal kinase

kDa

Kilodalton

LPS

Lipopolysaccharides

LRRs

Leucine-rich repeats

Man

Mannose

MAPK

Mitogen-activated protein kinase

MD-2

Myeloid differentiation factor 2

MHC

Major histocompatibility complex

MLNs

Mesenteric lymph nodes

MW

Molecular weight

NF-kB

Nuclear factor kappa B

NK cells

Natural killer cells

NO

Nitric oxide

p38

Protein 38

PI3K

1-phosphatidylinositol 3-kinase

PMA

Phorbol 12-myristate 13-acetate

polySia

Polysialic acid

PSA

Polysialic acid

Rha

Rhamnose

SA

Sialic acid

TCR

T cell receptor

Th

Helper T cell

TLRs

Toll like receptors

TNF

Tumor necrosis factor

Xyl

Xylose

ABSTRACT Toll-like receptor (TLR) 4 is an important polysaccharide receptor; however, the relationships between the structures and biological activities of TLR4 and polysaccharides remain unknown. Many recent findings have revealed the primary structure of TLR4/MD2-related

polysaccharides,

and

several

three-dimensional

structure

models

of

polysaccharide-binding proteins have been reported; and these models provide insights into the mechanisms through which polysaccharides interact with TLR4. In this review, we first discuss the origins of polysaccharides related to TLR4, including polysaccharides from higher plants, fungi, bacteria, algae, and animals. We then briefly describe the glucosidic bond types of TLR4-related heteroglycans and homoglycans and describe the typical molecular weights of TLR4-related polysaccharides. The primary structures and activity relationships of polysaccharides with TLR4/MD-2 are also discussed. Finally, based on the existing interaction models of LPS with TLR4/MD-2 and linear polysaccharides with proteins, we provide insights into the possible interaction models of polysaccharide ligands with TLR4/MD-2. To our knowledge, this review is the first to summarize the primary structures and activity relationships of TLR4-related polysaccharides and the possible mechanisms of interaction for TLR4 and TLR4-related polysaccharides.

Key words: Polysaccharide; Toll-like receptor 4; homoglycan; heteroglycan; glucosidic bond; molecular weight

1. Introduction Toll-like receptor (TLR) 4, in the form of TLR4/MD-2 complexes, is a cell surface receptor that associates with CD14 and mediates phagocytic inflammatory responses to a variety of microbes by activating nuclear translocation and the pro-inflammatory response (Lee et al., 2004; Zughaier, 2011). TLR4 was discovered early in 1998 (Kirschning, Wesche, Ayres & Rothe, 1998; Poltorak et al., 1998; Rock, Hardiman, Timans, Kastelein & Bazan, 1998) and has been extensively studied as a potential drug target in cancer, rheumatoid arthritis, septicaemia, and allergic asthma (Duez, Gosset & Tonnel, 2006; Rezaei, 2006). However, all TLR4 antagonists and agonists have shown poor results in clinical studies, for example, Eritoran (E5564) was not effective in septicemia (Connolly & O'Neill, 2012). Polysaccharides can function as biological response modifiers (Hironaka, Yamaguchi, Okita, Okawaki & Nagamine, 2006; Willment, Gordon & Brown, 2001; Zhang et al., 2013b), and TLR4 has been shown to function as an important polysaccharide receptor. Indeed, studies have shown that many polysaccharides are potent TLR4 agonists; these polysaccharides are widely distributed in higher plants, algae, microorganisms, and animals (Kim et al., 2013; Maue et al., 2013; Schepetkin, Kouakou, Yapi, Kirpotina, Jutila & Quinn, 2013). Additionally, polysaccharides an important component of various traditional Chinese medicines (TCMs) and have efficacy with low toxicity (Hou et al., 2013;

Liu et al., 2013; Peng, Li, Feng, Chen, Xu & Hu, 2013; Xiao et al., 2013; Zhao et al., 2014). Polysaccharides also possess important regulatory activities, mediating inflammation, aging, cancer, pathogen infection, and other physiological and pathological processes (Evrard et al., 2010; Hwang, Ahn, Park, Ha, Song & Jee, 2011; Udan, Ajit, Crouse & Nichols, 2008). However, it is difficult to determine the higher order structures of polysaccharides; therefore, determination of polysaccharide receptor structure-activity relationships is also challenging. Various factors, including monosaccharide composition, glucosidal bonds, molecular size, branching degree, and the overall molecular conformation, may influence polysaccharide activity (Chandarajoti, Xu, Sparkenbaugh, Key, Pawlinski & Liu, 2014; Liu et al., 2014; Zhang et al., 2014c). Although few higher order structures of TLR4-related polysaccharides have been elucidated, some studies have shown

definite

activity

for

higher

plant

ginseng

polysaccharides,

mushroom

polysaccharides, algal bladder wrack, and animal chitin (Kim et al., 2010a; Koller, MuellerWiefel, Rupec, Korting & Ruzicka, 2011; Rinaudo, 2006; Tominaga et al., 2010; Wilson, Wong, Stryjecki, De Boer, Lui & Mutch, 2013; Yoshida et al., 2012). Additionally, the TLR4/MD-2-lipopolysaccharide (LPS) complex crystal structure was determined at 3.1-Å resolution, and some line polysaccharide-lectins interaction models have been developed (Bricheux et al., 2013). Moreover, the primary structures of many TLR4-related polysaccharides have been determined, and these results may provide insights into future studies of polysaccharide structure-activity relationships. However, the primary structure and activity relationships of TLR4-related polysaccharides are still not well understood. This review discusses the various origins, structures, interactions, and molecular

weights of TLR4-related polysaccharides and summarizes the primary structure and activity relationships of TLR4 and related polysaccharides. We also provide perspectives on potential interaction models of polysaccharide ligands and the TLR4/MD-2 complex. 2. Monosaccharide composition More than 30 types of active polysaccharides can interact directly or indirectly with TLR4. The higher order structures of these polysaccharides are still unknown; however, recent studies have provided new evidence for their primary structures. The monosaccharide compositions of polysaccharides from different sources vary substantially but also have common features. For example, although there are hundreds of natural polysaccharides, there are few categories of monosaccharides; these primarily include glucose, galactose, arabinose, mannose, galacturonic acid, glucuronic acid, Nacetylgalactosamine, N-acetylglucosamine, xylose, fructose, rhamnose, fucose, sialic acid, and other derivatives. 2.1. Higher plant polysaccharides At least 11 higher plant polysaccharides can interact directly or indirectly with TLR4 (Table 1). The main polysaccharide genus categories include the following: Apple polysaccharide (AP) from apples in the Rosaceae family (Li et al., 2010; Zhang et al., 2012b); safflower polysaccharide fraction 1/2 (SF1/SF2) and inulin from Carthamus tinctorius L. and inulin from the Compositae family (Ando et al., 2002; Nagahara et al., 2011); angelan and angelica sinensis polysaccharide (AAP) from Angelica gigas Nakai and Angelica dahurica of the Apiaceae family (Chen, Duan, Qian, Guo, Song & Yang, 2010; Kim et al., 2007; Yang, Zhao, Wang & Mei, 2007b); astragalus polysaccharide (APS) from

Astragalus mongholicus of the Fabaceae family (Liu, Yao, Yu, Dong & Sheng, 2011b); arabinogalactan polysaccharide (G1-4A) from Tinospora cordifolia of the Menispermaceae family (Raghu, Sharma, Ramakrishnan, Khanam, Chintalwar & Sainis, 2009); lycium barbarum polysaccharide fraction 4 (LBPF4-OL) from Lycium barbarum L. of the Solanaceae family (Zhang et al., 2014a; Zhang et al., 2011); bioactive polysaccharides (named ZPF1) from Dioscorea batatas of the Dioscoreaceae family (Liu et al., 2008b); rhamnogalacturonan II (RG-II) from ginseng of the Araliaceae family (Ahn, Song, Yun, Jeong & Choi, 2006); and the Platycodon grandiflorum (PG) polysaccharide from Platycodon grandiflorum of the Campanulaceae family (Yoon et al., 2003; Yoon, Kang, Han, Park, Lee & Kim, 2004). Of the 11 categories, five are of acidic polysaccharides: AP from apples, AAP from Angelica sinensis, APS from the Astragalus membranaceus (Huangqi) dried root, G1-4A from Tinospora cordifolia, and angelan from Angelica gigas Nakai. The other six are all neutral polysaccharides. From the different genera mentioned above, galactose is produced by the greatest number of genera (9/11), followed by glucose (6/11), arabinose (6/11), rhamnose (6/11), galacturonic acid (5/11), mannose (4/11), xylose (2/11), fructose (2/11), and glucuronic acid (1/11). Additionally, N-acetylgalactosamine, Nacetylglucosamine, fucose, and sialic acid were not reported (Fig. 1A). 2.2. Fungal polysaccharides Fungal polysaccharides are important TLR4-related polysaccharides, and at least 10 fungal polysaccharides can activate TLR4 (Table 2). The main categories of fungal polysaccharides include Reishi-F3 from Reishi of the Polyporaceae family (Hsu et al., 2009; Yeh, Chen, Yang, Chuang & Sheu, 2010) ; cordlan from Cordyceps militaris of the

Clavicipitaceae family (Park, Hayashi & Park, 2012); cell wall polysaccharides from Aspergillus fumigatus (AFPS) of the Moniliaceae family (Chai et al., 2011); polyporus polysaccharide (PPS) from Polyporus umbellatus of the Polyporaceae family (Li & Xu, 2011; Li, Xu & Chen, 2010); protein bound polysaccharide K (PSK) from Coriolus versicolor of the Tulostomataceae family (Wang, Dong, Tan, Yu & Bao, 2013); zymosan from yeast of the Saccharomycetaceae family (Yang, Liu, Liu, Zhang & Guan, 2010a); yancheng polysaccharide (YCP) from Phoma herbarum YS4108 of the Sphaeropsicaceae family (Zhang et al., 2013b); glucuronoxylomannan (GXM) from cryptococcus neoformans of the Saccharomycetaeae family; sparan from Sparassis crispa of the Sparassidaceae family (Kim et al., 2010b); and mannan from Saccharomyces cerevisiae yeasts. Of the 10 categories of fungal polysaccharides listed above, two are acidic polysaccharides, including cordlan of Cordyceps militaris and GXM of Cryptococcus neoformans. Two glucidamins were noted, including Reishi-F3 of Ganoderma lucidum and the Aspergillus cell wall polysaccharide of Aspergillus fumigatus. The other six polysaccharides are neutral polysaccharides. Among these 10 different polysaccharide categories, glucose was the most frequent monosaccharide component (7/10), followed by mannose (6/10), galactose (4/10), fucose (2/10), xylose (2/10), rhamnose (1/10), arabinose (1/10), galacturonic acid (1/10), glucuronic acid (1/10), N-acetylgalactosamine (1/10), and N-acetylglucosamine (1/10). Notably, fructose and sialic acid were not reported (Fig. 1B). 2.3. Bacterial polysaccharides Bacterial polysaccharides are another important TLR4-related polysaccharide type, and at least five bacterial polysaccharides can activate or inhibit TLR4 (Table 3). The main

bacterial polysaccharide categories include polySia from Neisseria meningitidis (Zughaier, Svoboda, Pohl, Stephens & Shafer, 2010); the capsule polysaccharide of the Klebsiella pneumoniae（KCPS） family (Frank et al., 2013); the Vi capsular polysaccharide of Salmonella enterica serotype Typhi (Jansen et al., 2011); the Acetobacter polysaccharide 1 (AC-1) of the Acetobacter xylinum family; and xanthan gum (XG) of the Xanthomonas campestris pathovars family (Takeuchi et al., 2009). From the five bacterial polysaccharide categories listed above, three are acidic polysaccharides, including acidic polysaccharides from Klebsiella pneumoniae, Xanthomonas campestris and Neisseria meningitides. The Vi capsular polysaccharide of Salmonella enterica serotype Typhi is a glucidamin. Of these different polysaccharides, glucose was the most frequent monosaccharide (3/5), followed by glucuronic acid (2/5), mannose (1/5), fucose (1/5), N-acetylgalactosamine (1/5), and sialic acid (1/5). Additionally, galactose, rhamnose, arabinose, xylose, galacturonic acid, fructose, and N-acetylglucosamine were not reported (Fig. 1C). 2.4. Algal and animal polysaccharides Polysaccharides isolated from algae have been reported to enhance the phagocytic and secretory activity of macrophages and induce production of reactive oxygen species (ROS), nitric oxide (NO), and cytokines (e.g., tumor necrosis factor [TNF]-α, interleukin [IL]1, and IL-6). There are nine algae species from five divisions, including Aphanizomenon flos-aquae

(Nostocaceae),

Chlorella

pyrenoidosa

(Chlorellaceae),

Cladosiphon

okamuranus ToKida (Chordariaceae), Euglena gracilis (Euglenaceae), Gracilaria verrucosa (Gracilariaceae), Hizikia fusiformis (Sargassaceae), Porphyra yezoensis (Bangiaceae),

Sargassum

thunbergii

(Sargassaceae),

and

Spirulina

platensis

(Pseudanabaenaceae) (Schepetkin & Quinn, 2006). The main TLR4-related algae polysaccharide categories include hot-water-soluble polysaccharides from Chlorella pyrenoidosa (CWSP) of the Chlorophyta family (Hsu, Jeyashoke, Yeh, Song, Hua & Chao, 2010a); acetyl fucoidan (CAF) from Cladosiphon okamuranus (Teruya, Tatemoto, Konishi & Tako, 2009); and carrageenan from red algae (Bhattacharyya et al., 2010) (Table 3). CAF from Cladosiphon okamuranus (brown alga) contains glucuronic acid, and the other algae origin polysaccharides are neutral polysaccharides. Arabinose, galacturonic acid, fructose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid were not reported from these polysaccharide sources. Additionally, only one animal polysaccharide, lacto-Nfucopentaose III (12–25 molecules)-dextran (LNFPIII-Dex) of Taenia crassiceps, can activate the TLR4 signaling pathway (Fig. 1D).

Table 1 Monosaccharide compositions of TLR4-related polysaccharides from higher plants. Monosaccharide compositions Genus

Polysaccharide Glc

Malus

AP

Angelica

AAP

Carthamus

SF1/SF2

Astragalus

APS

Tinospora

G1-4A

Angelica gigas

Angelan

Lycium

LBPF4-OL

Dioscorea

ZPF1

Chrysanthemum

Inulin

Panax ginseng

RG-II

Platycodon

PG

Gal

Man

Rha

Ara

Xyl

GalA

GlcA

Fuc

Fru

GalNAc

GlcNAc

SA

Table 2 Monosaccharide composition of TLR4-related polysaccharides from fungi. Monosaccharide compositions Genus

Polysaccharide name Glc Gal

Ganoderma lucidum

Reishi-F3

Aspergillus fumigatus

AFPS

Cordyceps militaris

Cordlan

Cryptococcus neoformans

GXM

Polyporus umbellatus

PPS

Coriolus versicolor

PSK

Saccharomyces

Zymosan

Saccharomyces cerevisiae yeasts

Mannan

Phoma herbarum YS4108

YCP

Sparassis crispa

Sparan

Man

Rha

Ara

Xyl

GalA

GlcA

Fuc

Fru GalNAc

GlcNAc

SA

Table 3 Monosaccharide compositions of TLR4-related polysaccharides from bacteria, algae, and animals. Monosaccharide compositions Genus

Polysaccharide name Glc

Klebsiella pneumoniae

KCPS

Xanthomonas campestris

Xanthan gum (XG)

Neisseria meningitidis

PolySia

Salmonella enterica

SViCPS

Acetobacter xylinum

AC-1

Chlorella pyrenoidosa

CWSP

Cladosiphon okamuranus

CAF

Red algae

Carrageenan

Taenia crassiceps

LNFPIII-Dex

Gal

Man

Rha

Ara

Xyl

GalA

GlcA

Fuc

Fru GalNAc

GlcNAc

SA

Fig. 1. Frequencies of various monosaccharides in TLR4-related polysaccharides from difference sources. A, Monosaccharide frequencies in polysaccharides from higher plants. B, Monosaccharide frequencies in polysaccharides from fungi. C, Monosaccharide frequencies in polysaccharides from bacteria. D, Monosaccharide frequencies in polysaccharides from algae and animals.

3. Heteroglycan glucosidic bonds 3.1. Polymers composed of two monosaccharaide types 3.1.1. Glucose and fucose polymers PSK: PSK was isolated from the CM-101 Coriolus versicolor mushroom strain. PSK has a mean molecular weight (MW) of 94 kDa and is mainly composed of protein and glucan, which has a β-(1,4) bond in the main chain and β-(1,3) and β-(1,6) bonds in the side chain that binds to protein moieties through O- or N-glycosidic bonds. PSK has been shown to be effective in the treatment of various allogeneic and syngeneic animal tumors and has been given orally to patients with cancer (Hoshi et al., 2011; Maehara et al., 2012). Recent studies have found that multiple human pancreatic adenocarcinoma cells can be protected from PSK-mediated growth inhibition by neutralizing antibodies against TLR2 and TLR4. Additionally, a significant growth inhibition and additive effect was observed with PSK and gemcitabine when they were administered as combined treatment (Rosendahl, Sun, Wu & Andersson, 2012). ). In an in vitro research study, pre-incubation with TLR4 blocking antibody inhibited PSK-induced TNF-α secretion by both macrophages (J774A.1) and primary splenocytes. PSK promoted TNF-α and IL-6 secretion by wild-type but not TLR4-deficient peritoneal macrophages. These results indicated that PSK acts as a ligand for TLR4 receptors, leading to induction of inflammatory cytokines, such as TNF-α and IL6 (Price, Wenner, Sloper, Slaton & Novack, 2010). LNFPIII-Dex: LNFPIII-Dex activates dendritic cells (DCs) via TLR4, as does LPS. However, unlike LPS, LNFPIII-Dex-activated cells induce T helper 2 (Th2)-type CD4+ Tcell responses. LNFPIII-activated antigen-presenting cells (APCs) differentially activate nuclear factor kappa B (NF-κB). In contrast to LPS, LNFPIII-stimulated APCs that only transiently activate NF do not induce degradation of known inhibitor of κB (IκB) family members or production of NO. Cells stimulated with LNFPIII rapidly accumulate p50, suggesting that an alternative p105 degradation-dependent mechanism downstream of LNFPIII is primarily responsible for NF-kB activation (Thomas, Carter, Da'dara, DeSimone & Harn, 2005).

3.1.2. Polymers of glucose and fructose Inulin: Inulin is a nonstarch polysaccharide consisting of fructose unit chains that are coupled by β-(2,1) bonds and frequently terminated by a single glucose moiety, which occurs naturally as a storage carbohydrate in many plant species (Havenaar, BonninMarol, Van Dokkum, Petitet & Schaafsma, 1999). Specifically, inulin is a polydisperse polysaccharide consisting mainly, if not exclusively, of β-(2,1) fructosyl fructose units (Fm) with one glucopyranose unit at the reducing end (GFn). Fructose molecules in the GFn form are all present in the furanose form. However, in the Fm form, the ending and reducing fructose is in the pyranose form. Additionally, inulin is a slightly branched fructan; in inulin from chicory and dahlia, β-(2,6)-branches are present in proportions of 1–2% and 4–5%, respectively (Dan, Ghosh & Moulik, 2009; Kelly, 2008; Stevens, Meriggi & Booten, 2001). The chain lengths vary between 2 and 65 fructose units. Like other polysaccharides, inulin possesses a single helical conformation in solution. Inulin can enhance various immune responses, mainly to T and B cells, natural killer cells, and macrophages, in vivo and in vitro. Moreover, inulin has a direct effect on phorbol 12-myristate 13-acetate-treated THP1 macrophages. Inulin-induced phagocytosis is suppressed in peritoneal macrophages from TLR4-mutant C3H/HeJ mice. Moreover, inulin-induced TNF-α secretion from THP-1 macrophages is inhibited when using a blocking antibody specific for TLR4, suggesting that TLR4 is involved in inulin binding to macrophages. Furthermore, an inulin-induced phagocytosis test revealed that phosphoinositide 3-kinase and mitogen-activated protein kinase, particularly p38, participate in phagocytosis. These results suggested that inulin may affect macrophages directly through the TLR4 signaling pathway and stimulation of phagocytosis to enhance immunomodulation (Nagahara et al., 2011). 3.1.3. Glucose and mannose polymers Zymosan is a potent inflammatory substance that has immunopharmacological activity and was isolated from the yeast Saccharomyces cerevisiae. The major component of zymosan is β-glucan; however, zymosan also contains other constituents, such as mannan, protein, and nucleic acids. Sodium hypochlorite (NaClO) treatment of zymosan can lead to formation of oxidized zymosan. Nuclear magnetic resonance (NMR) analysis

of a native oxidized zymosan and zymolyase (endo-1,3-β-glucanase) digestion revealed that oxidized zymosan contains 1,3-β-linked and 1,6-β-linked glucan moieties, the latter of which is degraded by sodium metaperiodate treatment. The polymerization degree (DP) of the 1,6-β-glucan moiety was estimated to be about DP10-DP50 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis. In a rat model of multiple organ dysfunction syndrome (MODS), when adult Wistar rats were challenged intraperitoneally with zymosan, they developed biochemical and histological abnormalities similar to those observed in human MODS as compared with controls. Additionally, zymosan challenge resulted in an increase in TLR4 mRNA expression in all three organs tested (Yang, Liu, Liu, Zhang & Guan, 2010b). However, in another study, zymosan prepared from the fungal wall was shown to be independent of TLR, complement, and dectin-1 (Kelly et al., 2008). The structure and activity relationships of zymosan were also observed, including zymosan subjected to sodium metaperiodate oxidation (NaIO4) and borohydride reduction (I/B-zymosan) and/or limited hydrolysis of oxidized moieties (I/B/H-zymosan). As a general observation, NaIO4, oxidation products exhibited reduced, but still significant, activity. The H2O2 production induced by I/B/H-zymosan was significantly reduced after extensive sonication. Antagonist(s) for H2O2 synthesis were concomitantly solubilized by sonication of I/B/H-zymosan. In contrast, TNF-α production induced by I/B/H-zymosan was comparable to that of zymosan. These observations strongly suggest that highly branched 1,3-β- and 1,6-β-glucosidic linkages resistant to NaIO4 oxidation are important for the biological activity of zymosan. Furthermore, the minimal structure in zymosan necessary for biological activity may depend on the activity tested. The active moiety of zymosan was also fractionated by its solubility in water, yielding water-soluble (ZWS) and water-insoluble (ZWIS) fractions, and its biological activity toward macrophages and TLR transfectants was examined. ZWS showed higher induction of TNF-α production. Moreover, NF-κB activation via TLR2, TLR1/TLR2, TLR2/TLR6, and TLR4/MD-2/CD14 was also enhanced by stimulation with ZWS and ZWIS. In particular, ZWS showed higher activity via TLR1/TLR2, TLR2/TLR6, and

TLR4/MD-2/CD14 than other preparations. ZWS activity was decreased by treatment with polymyxin B, but not with lysozyme or zymolyase. 3.1.4. Galactose and rhamnose polymers RG-II: RG-II is a component of ginseng and grapes (Shin, Kiyohara, Matsumoto & Yamada, 1997, 1998), that has unusual anti-tumour response activity via immune enhancement in a different manner than resveratrol. RG-II has also been isolated from the cell walls of sycamore (Acer pseudoplatanus), Douglas fir ( Pseudotsuga menziesii), rice ( Oryza sativa), onion (Allium cepa), and kiwi fruit (Actinidia deliciosa) and is present in cultured sycamore cells medium, in the commercial enzyme preparation of Pectinol AC, and in red wine (Whitcombe, Oneill, Steffan, Albersheim & Darvill, 1995). RG-II is a lowmolecular-mass polysaccharide (5-10 kDa) that was first identified in 1978 as a polysaccharide complex that is solubilized with an endopolygalacturonase treatment of suspension-cultured sycamore cell walls. RG-II is a pectic polysaccharide, which is referred to as a substituted galacturonan, and has a backbone that is composed of linear α-(1,4)-D-GalpA residues (O'Neill, Ishii, Albersheim & Darvill, 2004). Some results suggested that RG-II expedites its dendritic cell-based immune response through the TLR4 signalling pathway. The effectiveness of an RG-II-stimulated bone marrow-derived dendritic cell (BMDC) vaccination was observed in the induction of anti-tumour immunity in a mouse lymphoma model using EG7-lymphoma cells expressing ovalbumin (OVA). In a BMDC study from mice that were deficient in TLRs, results showed that RG-II activity was dependent on TLR4. RG-II showed a preventive effect of immunization with OVApulsed BMDCs against EG7 lymphoma (Park et al., 2013). Additionally, another RG-II polysaccharide, KMPS-2E (Mw 84.8 kDa), was found to inhibit TLR4 expression in macrophages. The backbone of KMPS-2E consists [-6)-β-D-Galp(1-3)-β-L- Rhap-(1-4)-βD-GalpA-(1-3)-β-D-Galp-(1-] units with the 5)-β-D –Arap(1-3,5)-β-D-Arap (1-attached to the backbone through O-4 of (1-3,4)-L-Rhap) side chain. T-β-D-Galp is attached to the backbone through O-6 of the (1-3, 6)-β-D-Galp residues and T-β-D-Ara is connected to the end group of each chain. KMPS-2E (50, 100 and 200 μg/mL) inhibits iNOS, TLR4, phospho-NF-kB-p65 expression, phosphor-IKK, phosphor-IkB-α expression as well as IkB-

ɑ degradation and inflammatory cytokine gene expression (TNF-α, IL-1β, iNOS and IL-6) that is mediated by the NF-kB signalling pathway in macrophages (Li et al., 2014). 3.2. Polymers containing three types of monosaccharides 3.2.1. Glucose, galactose, and mannose polymers ZPF1: The yam (Dioscorea) has been widely used to improve health and for the treatment of several illnesses in traditional medicine (Farombi, Britton & Emerole, 2000; Hsu, Wu, Liu & Cheng, 2007). The major constituents of yams, which include polysaccharides and proteins, were found to have antioxidant, immunostimulatory, and angiotensin converting enzyme inhibitory activities (Choi & Hwang, 2002; Choi, Koo & Hwang, 2004; Hsu, Lin, Lee, Lin & Hou, 2002). The bioactive polysaccharides (named ZPF1) from yam (Dioscorea batatas) were chemically determined. This study found that repeating β-1,4-mannan residues exhibited acetylation on C-2-OH and C-3-OH for approximately 28% of the structures. The major sugar component of ZPF1 is mannose (76.7%), and ZPF1 also contains glucose and galactose (16.9% and 6.3%, respectively). The ZPF1 sugar linkage is mainly 1,4-mannosidic, and the anomeric configuration was assigned as the β-form due to the finding of a 1JH-C of 160 Hz in the 1H NMR spectrum (Liu et al., 2008a). ZPF1 participates in the stimulation of murine wild-type macrophages, leading to TNF-α production. Moreover, TLR4 has been shown to be involved in ZPF1mediated TNF-α secretion (Liu et al., 2008a). PPS: PPS was isolated from the boiling water extract of Zhuling, followed by ethanol precipitation, dialysis, and protein depletion using the Sevag method. The resulting polysaccharide extract was dialyzed against water and then lyophilized. The PPS molecules had a MW of approximately 160 kDa, and the molecular weight distribution (Mr/Mn) was 2.914. Additionally, PPS was mainly composed of D-glucose units but also contained D-galactose and D-mannose. The structure of PPS was determined with Fourier transform infrared spectroscopy (FTIR); the results showed that PPS had an absorption peak characteristic of a β-D-glucopyranoside. Treatment of bone marrow-derived dendritic cells (BMDCs) with PPS resulted in enhanced CD86 cell-surface expression and increased production of both IL-12 p40 and IL-10, in a concentration-dependent manner. PPS-

induced IL-12 p40 production was inhibited by monoclonal antibodies targeting TLR4. Additionally, flow cytometric analysis showed that fluorescence-labeled PPS (f-PPS) bound specifically to BMDCs. This binding was blocked by both unlabeled PPS and antiTLR4 monoclonal antibodies, but not by anti-TLR2 or anti-CR3 monoclonal antibodies (Li & Xu, 2011). The data show that PPS promoted the activation and maturation of murine BMDCs and macrophages via TLR4. AFPS: AFPS show differential capacities for modulation of host TLR-mediated IL-6 production. Beta-glucan specifically suppresses TLR4-induced responses, whereas αglucan inhibits TLR2- or TLR4-induced IL-6 production. Galactomannan blocks the TLR4mediated response but has limited effects on TLR2 signaling. Chitin, on the other hand, does not have significant immunomodulatory effects. The ability of the fungal cell wall to alter the immune signature of the pathogen may contribute to its virulence and co-infection pathogenesis (Chai et al., 2011). 3.2.2. Glucose, glucose aldehyde acid, and mannose polymers XG: XG is an exocellular biopolymer that is secreted by Xanthomonas sp. and has a β-D-(1,4)-linked glucan backbone with short trisaccharide side chains consisting of α-Dmannose, β-D-glucuronic acid, and β-D-mannose on alternating glucose residues (Jansson, Kenne & Lindberg, 1975). The main chain of XG is based on a linear backbone of 1,4-linked-β-D-glucose. At the C(3) position of every alternate glucose residue, there is a charged trisaccharide side chain containing a glucuronic acid residue between two mannose units. The mannose residue that links the side chain is acetylated, and the terminal mannose contains pyruvate groups at approximately every other side chain. The terminal β-D-mannose is linked at β-1,4 to the glucuronic acid which, in turn, is linked to α1,2 at the α-D-mannose. On approximately one-half of the terminal mannose residues, a pyruvic acid moiety is joined by a ketal linkage at the O(4) and O(6) positions. Acetate groups are present as substituents at the O(6) position of the nonterminal mannose. XG has been shown to undergo a thermally induced conformational change that is sensitive to the ionic strength, and there is still debate as to whether the adopted ordered structure involves single or double helices (Kitamura, Takeo, Kuge & Stokke, 1991; Milas, Reed &

Printz, 1996; Norton, Goodall, Frangou, Morris & Rees, 1984). In another study, XG was shown to stimulate macrophages in a myeloid differentiation primary response protein 88 (MyD88)-dependent manner and was mainly recognized by TLR4. Oral administration of XG significantly suppressed tumor growth and prolonged the survival of mice that were subcutaneously inoculated with B16Kb melanoma cells. Natural killer (NK) cell activity and tumor-specific cytotoxicity of CD8 T cells were augmented in XG-treated mice. Additionally, the in vivo anticancer effects of XG have also been shown to be dependent on TLR4. Additionally, the growth of syngeneic MBT-2 bladder tumors cells implanted in C3H/HeJ mice, which lack TLR4 signaling, is not affected by XG (Takeuchi et al., 2009). 3.2.3. Glucose, glucose aldehyde acid, and fucose polymers KCPS: KCPS was purified and its sugar composition was determined (Chung et al., 2007). The KCPS of the wild-type NTUH K-2044 and the wbbO mutant are identical, and they consist of fucose, glucose, and glucuronic acid in a 1:1:1 ratio. Chemical analyses, mass fragmentation, and NMR assignment of partial acid-treated CPS fragments revealed that the chemical structure of CPS was a trisaccharide repeating unit of (-3)-β-D-Glc-(1-4)[2,3-(S)-pyruvate]-β-D-GlcA-(1-4)-α- L-Fuc-(1-). Thus, the KCPS structure from the NTUHK2044 PLA K. pneumoniae strain is similar to that of the K1 K. pneumoniae strain (Zamze et al., 2002). In addition to the pyruvation modification of both K1 strains and NTUH K2044, the NTUH-K2044 strain PLA K. pneumoniae KCPS is highly acetylated. The glucuronic acid residue of the trisaccharide repeating unit was pyruvylated 48–54% of the time, which suggests that every other trisaccharide repeating unit is modified by pyruvylation. The O-acetylation position was determined from the reduced KCPS derivatives and further analysed by GC-MS, showing that 6% of the fucosidic linkages were 1,4-linked, as estimated on the basis of molar equivalence. Moreover, the 1,2,4-fucosidic linkage amounts were almost equal to that of the 1,3,4-fucosidic linkages, although no 1,2,3,4-fucosidic linkages were found. These results show that either the C2-OH or the C3OH of fucose, but not both, are acetylated and that the total acetylation level is ~94%, i.e., it is nearly stoichiometric (Yang et al., 2011). Pharmacology research found that PLA K. Pneumonia KCPS induces TNF-α and IL-6 secretion by macrophages through TLR4 and

that this effect was lost when pyruvation and O-acetylation were chemically destroyed. Furthermore, TNF-α and IL-6 expression in PLA K. pneumoniae CPS-stimulated macrophages was shown to be regulated by the TLR4/ROS/PKC-δ/NF-kB, TLR4/PI3kinase/AKT/NF-kB, and TLR4/MAPK signalling pathways (Regueiro, Moranta, Campos, Margareto, Garmendia & Bengoechea, 2009; Wu et al., 2009). 3.2.4. Glucose, galactose, and arabinose polymers APS: The immunopotentiating effect of the Astragalus membranaceus root, a medicinal herb, has been associated with its polysaccharide fractions. The APS polysaccharides are a mixture of APS I and II. APS I is a type of heterosaccharide that is composed of D-glucose, D-galactose, and L-arabinose in molar ratios of 1.75:1.63:1, with the average MW being 36.3 kDa. APS II is a type of dextran with a high MW that is bonded mainly with α-(1,4)-D-glycosidic linkages (Chen et al., 2012; Rogers et al., 1999). A molecular immunopotentiating mechanism study showed that a monoclonal Ab against mouse TLR4 partially inhibited APS binding by macrophages, implying that a direct interaction between APS and TLR4 on cell surface occurs (Shao, Xu, Dai, Tu, Li & Gao, 2004). Additionally, TLR4 expression in the bladder and macrophages of mice that received APS was higher than that in the controls in vivo and in vitro (Yin et al., 2010). APS is also a potent adjuvant for the hepatitis B subunit vaccine and can enhance both humoral and cellular immune responses via activating the TLR4 signaling pathway and inhibit the expression of TGF-β and frequency of Treg cells (Du et al., 2011). APS might suppress CD4+CD25+ Treg activity, at least in part, via binding TLR4 on Tregs and trigger a Th2 to Th1 shift with CD4+T cell activation in burned mice with P. aeruginosa infections (Liu, Yao, Yu, Dong & Sheng, 2011a). 3.2.5. Glucose aldehyde acid, mannose, and xylose polymers GXM: Cryptococcus neoformans is encapsulated yeast that is responsible for meningoencephalitis in individuals with suppressed immune systems. The yeast's virulence is partially due to its PS capsule. The major capsular antigen is a high MW glucuronoxylomannan (GXM) that is partially 6-O-acetylated on mannose (Bhattacharjee, Bennett & Glaudemans, 1984). Serological reactivity of the capsular polysaccharide has

traditionally been used to define four serotypes (A, B, C, and D) that have been classified into two varieties based on biochemical differences: var. neoformans (serotypes A and D) and var. gattii (serotypes B and C) (Bennett, Kwon-Chung & Howard, 1977; Bennett, KwonChung & Theodore, 1978; Kwon-Chung, Polacheck & Bennett, 1982). A simple structural relationship between the GXM polysaccharide of the four serotypes exists. They are composed of a core repeating unit, to which (1,2)-linked β-D-Xylp and (1,4)-linked β-D-Xylp residues are added to the (1,3)-linked α-D-mannopyrannan backbone. Homogeneous structural models were originally proposed for the four serotypes based on precise molar Xyl/Man/GlcA ratios of 1:3:1, 2:3:1, 3:3:1, and 4:3:1 for serotypes D, A, B, and C, respectively, with Xylp substitution at O-2 for the var. neoformans serotypes and Xylp substitution at O-2 and O-4 for the var. gattii serotypes (Bhattacharjee, Bennett & Glaudemans, 1984). Studies have identified CD14, CD18, TLR2 and TLR4 as putative receptors for GXM (Ellerbroek, Ulfman, Hoepelman & Coenjaerts, 2004; Monari et al., 2005; Monari, Pericolini, Bistoni, Casadevall, Kozel & Vecchiarelli, 2005; Yauch, Mansour & Levitz, 2003; Yauch, Mansour, Shoham, Rottman & Levitz, 2004). GXM binding to macrophage receptors triggers activation of NF-kB, but not MAPKs. 3.2.6. Glucose aldehyde acid, fucose, and xylose polymers Fucoidan: Fucoidan is a sulfated polysaccharide that is found in the cell wall matrix of brown algae. The acetyl fucoidan (CAF) structure consists of α-(1-3) linked L-fucosyl residues that are substituted with D-glucuronic acid at C-2 and sulfate groups at C-4 of the L-fucosyl residues. Neutralizing anti-TLR4, anti-CD14 and anti-scavenger receptor class A (SRA), but not anti-complement receptor type 3 monoclonal antibodies, decreased CAFinduced nitric oxide production. The results suggested that CAF induced macrophage activation through membrane receptors TLR4, CD14 and SRA (Teruya, Tatemoto, Konishi & Tako, 2009). 3.2.7. Galactose, galacturonic acid, and arabinose polymers Angelan: Angelan is a polysaccharide that is purified from the root and cell culture of Angelica gigas Nakai (Ahn, Sim, Kim, Han & Kim, 1998). Angelan (10 kDa MW ) was chemically characterized as a single substance, which was certified by the analytical high

performance liquid chromatography (HPLC) method. It is composed of a pectic polysaccharide, proteins, and other inorganic compounds (Ca2+ and Mg2+, etc.). The polysaccharide was composed of arabinose, galactose, and galacturonic acid, including uronic acid, but it did not contain glucose (Han et al., 1998; Jeon, Han, Ahn & Kim, 1999; Jeon & Kim, 2001). Angelan efficiently increased the maturation of TLR4 (+/+) dendritic cells from C5713L/6 and C3H/HeN mice, but not TLR4 (-/-) dendritic cells from C3H/ReJ mice. Phenotypic maturation was confirmed by elevated CD80, CD86, and MHC-class II molecule expression, and by functional maturation, which was demonstrated by increased IL-12 production, enhanced allogeneic T cell stimulation, and decreased endocytosis. These results indicate that angelan induces dendritic cell maturation via TLR4 signalling pathways and suggests the possible use of angelan in dendritic cell-based immunotherapies (Kimura, Sumiyoshi, Suzuki, Suzuki & Sakanaka, 2007). Additionally, clinical research found that angelan might be helpful in overcoming the disadvantages of dendritic cell-based cancer therapy, such as impaired maturation and poor migration in cancer patients (Kim et al., 2011). It was also reported that angelan could inhibit autoimmunity in non-obese diabetic (NOD) mice. Although 80% of the studied NOD mice developed diabetes by 24 weeks of age, none of the angelan-treated NOD mice developed diabetes. Histological examination of the pancreatic islets revealed that most of the islets isolated from the angelan-treated mice were less infiltrated with lymphocytes compared with those in the control mice. These results suggest that angelan has dual immunomodulatory functions, i.e., immunostimulation in tumour-bearing mice and immunosuppression in autoimmune diabetic mice (Kim et al., 2008). 3.2.8. Galactose, arabinose, and rhamnose polymers LBPF4-OL: Lycium barbarum L. is a renowned Yin strengthening agent in traditional Chinese medicine. LBPF4-OL is the glycan part of Lycium barbarum L. polysaccharide protein complex fraction 4 (LBPF4). Methylation analysis of LBPF4-OL indicated that a highly branching unit in LBPF4-OL was β-(1,4) Gal, and the major non-reducing end was Ara. Component analysis showed that LBPF4-OL was composed of Rha, Ara and Gal in a molar ratio of 0.05:1.33:1 (Zhang et al., 2011). LBPF4-OL can significantly induce TNF-α

and IL-1β production in peritoneal macrophages isolated from wild-type C3H/HeN but not TLR4-deficient mice C3H/HeJ (Zhang et al., 2014c). While L ruthenicum polysaccharide LRGP3 treatment significantly inhibited the LPS-induced NO production and the mRNA expression of iNOS, as well as the level of TLR4. Furthermore, LRGP3 treatment prevented the IΚB adegradation and reduced phospho-NF-B-kappa p65 protein expression in LPS-stimulated RAW264.7 cells. The results suggested that LRGP3 attenuated LPSinduced inflammation via inhibiting TLR4/NF-ΚB signaling pathway (Peng, Liu, Shi & Li, 2014). 3.3. Polymers with four monosaccharaide types 3.3.1. Galactose, arabinose, galacturonic acid, and rhamnose polymers G1-4A is a polysaccharide from an Indian medicinal plant Tinospora cordifolia. The mean MW of G1-4A, as determined by GPC analysis, is 2,200 kDa and the monosaccharide derivatives were identified and estimated by GC (Chintalwar et al., 1999). The major components of the polysaccharide are galactose (32%), arabinose (31%), galacturonic acid (35%) and rhamnose (1.4%). GC-MS analysis of the partially methylated alditol acetates, complete hydrolysis, NaBH4 reduction and acetylation showed the presence of a terminal arabinose, 1,5 linked arabinose, terminal galactose, 1,4-linked galactose, 1,6linked galactose and a 1,3,6-linked galactose. On the basis of these observations, G1-4A was characterized as an acidic arabinogalactan polysaccharide (Desai, Ramkrishnan, Chintalwar & Sainis, 2007; Pandey, Shankar & Sainis, 2012). The detailed molecular events associated with G1-4A induced immunomodulation in vitro and in vivo were revealed. An anti-TLR4/MD-2 complex antibody inhibited G1-4A induced B cell proliferation and IkB-α degradation suggesting that TLR4 was a receptor for G1-4A on B cells (Raghu, Sharma, Ramakrishnan, Khanam, Chintalwar & Sainis, 2009). 3.4. Polymers with five monosaccharaide types 3.4.1. Galactose, galacturonic acid, rhamnose, arabinose, and xylose polymers Cordlan: Cordyceps militaris is a representative insect-borne fungus that has been folklorically used. Various biological activities of Cordyceps militaris, such as anti-fibrotic, anti-inflammatory and anti-nociceptive activities have been reported (Nan, Park, Yang,

Song, Ko & Sohn, 2001; Won & Park, 2005). An acidic polysaccharide (Cordlan) was isolated from Cordyceps militaris extract grown on germinated soybeans. Sugar composition analyses indicated that Cordlan consisted of D-galactose, L-arabinose, Dxylose, L-rhamnose, and D-galacturonic acid. On the basis of methylation analysis results, cordlan was considered to be mainly composed of Araf-(1,5)-Araf-(1,4) -Galp-(1,4)-GalAp residues (Ohta, Lee, Hayashi, Fujita, Park & Hayashi, 2007). Cordlan induced TNF-α, IL12 p40, and IL-8 levels as well as TLR2 and TLR4 mRNA expression in THP-1 human monocytes. Cordlan was developed as a promising immunmodulating agent with macrophage-activating properties (Park, Hayashi & Park, 2012). Cordlan induced maturation of TLR4 (+/+) DCs from C3H/HeN mice, but not TLR4 (-/-) DCs from C3H/HeJ mice, suggesting a promising membrane receptor for Cordlan. Additionally, Cordlan increased ERK, p38, and JNK phosphorylation, and NF-ΚB p50/p65 nuclear translocation, which are the main signalling molecules that are down-stream of TLR4. These results indicate that Cordlan induces DC maturation through TLR4 signalling pathways (Kim et al., 2010a). 3.4.2. Glucose, galactose, mannose, rhamnose, and xylose polymers CWSP: Chlorella, unicellular green algae, has often been used for healthimprovement purposes, including as a hypertension treatment and for the modulation of human immune responses. A hot-water-soluble polysaccharide from Chlorella pyrenoidosa (CWSP) was observed to be primarily composed of rhamnose (31.8%), glucose (20.42%), galactose (13.28%), mannose (5.23%), and xylose (1.27%), as analysed by GC-MS (Hsu, Jeyashoke, Yeh, Song, Hua & Chao, 2010a). It was reported that a hot-water extract of Chlorella might elicit various beneficial pharmacological effects against cancers, bacterial infections, and viral replication (Merchant & Andre, 2001; Queiroz, Rodrigues, Bincoletto, Figueiredo & Malacrida, 2003; Tanaka et al., 1998). Research found that CWSP induced IL-1β secretion in macrophages via TLR4 mediated protein kinase signalling pathways. Additionally, CWSP also stimulated cell HLA-DA, -DB, and -DC, and HLA-DR, -DP, and DQ surface expression as well as co-stimulatory family molecule expression, such as CD80 and CD86 in macrophages. Furthermore, pre-injection of C57BL/6J mice with CWSP

increased LPS-induced TNF-α and IL-1β secretion into serum in vivo (Hsu, Jeyashoke, Yeh, Song, Hua & Chao, 2010a). 3.5. Polymers with six monosaccharaide types 3.5.1. Glucose, galactose, mannose, rhamnose, arabinose, and galacturonic acid polymers AAP: Angelica sinensis (Oliv.) Diels, called Danggui in China, is a well-known oriental herb belonging to the Umbelliferae family. It is customarily used for the treatment of women’s diseases (e.g., regulating menstruation, relieving pain, anaemia, and stimulation in blood circulation) (Liu, Zhang, You, Zeng, Guo & Wang, 2012; Wang, Zeng, Liu, Guo & Zhang, 2011). Extraction of a crude water-soluble polysaccharide yielded approximately 10.8% (w/w) of dried A. sinensis, which is a primrose yellow powder. It was composed mainly of galactose, glucose, arabinose, rhamnose, and mannose. The polysaccharides were eluted with NaCl solutions ranging from 0.2 to 0.6M, and they were found to be acidic polysaccharides, as galacturonic acid was found (35.38%, 58.27%) and they were conjunct with a certain amount of protein (3.62%, 2.53%). The monosaccharaides that were identified were primarily galactose, arabinose, and rhamnose, as well as glucose and mannose (Jin, Zhao, Huang, Xu & Shang, 2012). Research found that AAP was shown to strongly augment TLR4 mRNA expression and the pretreatment of macrophages with antiTLR4 antibody significantly blocked AAP-induced NO release and the increase of iNOS activity and TNF-α secretion (Yang, Zhao, Wang & Mei, 2007a).

3.5.2. Glucose, galactose, galacturonic acid, mannose, xylose, and fucose polymers Reishi-F3: Ganoderma lucidum, a native biosidiomycetous fungus (also known as Reishi or Ling-Zhi), has long been used as a traditional Chinese herbal medicine to prevent and treat various human diseases, such as bronchitis, hepatitis, hypertension, neoplasia, and immunological disorders for over 2 millennia (Lin & Zhang, 2004). The main carbohydrate compositions of Reishi-F3 are 7.1% L-fucose, 3.1% D-xylose, 15.1% D-Man, 13.5% D-Gal, 1.2% D-GalNac, and 58.1% D-Glc (Chen et al., 2004; Lin et al., 2006).

Research found that Reishi-F3 causes mouse splenic B cell activation and differentiation to IgM-secreting plasma cells, and the process depends on Reishi-F3-mediated induction of Blimp-1, a master regulator capable of triggering a cascade of gene expression changes during plasmacytic differentiation. In human peripheral B lymphocytes, although Reishi-F3 fails to induce their activation, it is able to enhance antibody secretion, which is associated with Blimp-1 mRNA induction. The function of Reishi-F3 depends on TLR4/TLR2 as neutralizing antibodies against TLR4/TLR2 blocked Reishi-F3-mediated induction of Blimp1 mRNA and Ig secretion. 3.5.3. Glucose, Glucuronic acid, galactose, Galacturonic acid, mannose, and xylose polymers AP: studies have shown that fruit and vegetable consumption can prevent cancer occurrence (Gosse et al., 2005). Certain components of apples have been shown to prevent cancer growth and impede cancer progression. A research study showed that the monosaccharide compositions of hot water extracted apple (AP) polysaccharides mainly were mannose, glucuronic acid, galacturonic acid, glucose, galactose and xylose by the HPLC method. Moreover, apple polysaccharides significantly inhibited the binding of FITCLPS in two human colorectal cancer (CRC) cell lines, HT-29 and SW620 (Zhang et al., 2012a). Additionally, the modified Fuji apple polysaccharide (MAP) competed with LPS for binding to TLR4 to reduce LPS-induced NF-κB expression and suppressed the nuclear translocation of NF-κB p65. MAP significantly decreased LPS-induced expression of TLR4, cyclooxygenase-2, matrix metallopeptidase 9 (MMP9), matrix metallopeptidase 2, inducible nitric oxide synthase, and prostaglandin E2, and it increased inhibitor of κBα and NF-κB p65 protein expression in the cytoplasm when it was given in combination with LPS. The results indicate that MAP suppressed LPS-induced migration and invasiveness of CRC cells by targeting the LPS/TLR4/NF-κB pathway (Zhang et al., 2013a). 3.5.4. Glucose, galactose, mannose, rhamnose, arabinose, and xylose polymers SF1/SF2: the Safflower polysaccharide, SF1/SF2, was purified from dried safflower petals (Wakabayashi, Hirokawa, Yamauchi, Kataoka, Woo & Nagai, 1997). The gel filtration profiles revealed that the SF1 and SF2 MWs were estimated to be more than 100

kDa, and SF1 eluted faster than SF2. The main components of SF1 and SF2 are PS, which consists of neutral sugars, e.g., glucose, galactose, arabinose, xylose, rhamnose, and mannose. There is no significant difference in the sugar components, except that SF1 and SF2 contain more glucose and more arabinose, respectively. SF1 and SF2, which were purified from dried safflower petals (Carthamus tinctorius L.), stimulated the synthesis of various cytokines in peritoneal macrophages. Enforced expression of TLR4 and MD-2 rendered the responsiveness to SF1 and SF2. Moreover, this safflower polysaccharide failed to induce the production of TNF-α and NO by peritoneal macrophages that were prepared from C3H/HeJ mice that had a point mutation in the TLR4 gene. These observations clearly indicate that safflower polysaccharides activate the NF-kB signalling pathway via TLR4 (Ando et al., 2002).

4. Homoglycan glucosidic bonds 4.1. Glucan A variety of receptors, including lactosylceramide (LacCer), TLR2, TLR6, and dectin1, are closely related to β-D-glucan activity (Brown & Gordon, 2001; Gantner, Simmons, Canavera, Akira & Underhill, 2003; Young, Ye, Frazer, Shi & Castranova, 2001). ). However, other studies have reported that microbial sources of glucans, including the Phoma herbarum YS4108 glucan YCP, Aureobasidium sp BRD-glucan, and Sparassis crispa glucan sparan polysaccharides, are closely associated with TLR4. Additionally, the animal source of chitin and hyaluronic acid activity is associated with TLR4 (Termeer et al., 2002). There are four types of glucan glycosidic bonds: β-(1,3) (Barsanti, Passarelli, Evangelista, Frassanito & Gualtieri, 2011; Harada et al., 2006b), β-(1,4), α-(1,4), and β(1,6) glycosidic bonds (Bae et al., 2013; Harada, Miura, Adachi, Nakajima, Yadomae & Ohno, 2003; Smiderle, Alquini, Tadra-Sfeir, Iacomini, Wichers & Van Griensven, 2013) (Table 4). Reports have shown that β-(1,3)-, β-(1,4)-, and α-(1,4)-linked glucan activity is related to TLR4. However, whether β-(1,6)-linked glucan activity is also related to TLR4 has not been reported.

4.1.1. ß-(1,3)-D-Glucan polymers Sparan: β-(1,3)-D-glucans are major structural components of fungal cell walls that modulate innate immunity in part by activation of macrophages in mammals (Harada et al., 2006a; Harada, Miura, Adachi, Nakajima, Yadomae & Ohno, 2004). Sparassis crispa is a medicinal mushroom containing high levels of six-branched β-(1,3)-D-glucan (sparan), which has one branch at every third main chain unit and exhibits immune-mediated anticancer activity. Research has shown that sparan significantly induces DC maturation by elevating the expression of CD40, CD80, CD86, MHC-I/II, IL-12, IL-1β, TNF-α, and interferon (IFN)-α/β; enhancing IL-2 production and proliferation in allogenic T cells; and decreasing endocytosis. TLR4 functions as a sparan membrane receptor, as demonstrated by impaired DC maturation in TLR4-knockout and TLR4-mutated C3H/HeJ mouse bone marrow cells and by the use of anti-TLR4/MD-2 neutralizing antibodies. Sparan increases the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun Nterminal kinase (JNK) and enhances NF-κB p50/p65 nuclear translocation in DCs. These results indicate that six-branched 1,3-β-D-glucan can activate DCs through TLR4 via the mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways. 4.1.2. ß-(1,4)-D-Glucan polymers AC-1: β-(1,4)-D-Glucan is the predominant polysaccharide in plant cell walls but is also produced by fungi and bacteria, such as Acetobacter species. AC-1 is a bacterial cellulose that is produced by Acetobacter xylinum and is composed of β-(1,4)-D-glucan with glucosyl residue branches. AC-1 has a molecular mass of 1,000 kDa and exhibits a strong capacity to induce IL-12 p40 and TNF-α production by macrophages in vitro via TLR4 signaling. The effects of AC-1 on antilisterial activity were found to be reduced in C3H/HeJ mice carrying mutated TLR-4. Thus, AC-1, a potent IL-12 inducer that functions through TLR-4, enhances protective immunity against L. monocytogenes via augmentation of Th1 responses. 4.1.3. α-(1,4)-D-glucan polymers YCP: Many studies have described the immunostimulatory properties of β-glucans (Kimura, Sumiyoshi, Suzuki & Sakanaka, 2006; Yamanaka et al., 2012), however, α-

glucans have not been well studied. YCP is a polysaccharide that has a mean MW of 2,400 kDa and was purified from the mycelium of Phoma herbarum YS4108, a marine filamentous fungus that inhabits sediment at a depth of 50 m in the Yellow Sea near Sheyang Port, Yancheng, China. YCP is water soluble, and its backbone likely possesses a linear α-(1,4)-linked glucose main chain with an α-(1,6)-linked side chain (Yang, Gao, Han, Xu, Song & Tan, 2005). YCP stimulates NO release in macrophages in vitro, and fluorescence-labeled YCP (fl-YCP) can bind directly to receptors on the surface of macrophages in a time- and concentration-dependent manner. Competition studies have shown that LPS, laminarin, anti-TLR4 antibodies, and anti-CD11b (CR3) antibodies could inhibit fl-YCP binding to macrophages and B lymphocytes. 4.2. Galactan Studies have shown that the activity of galactose polymer (hyaluronic acid) isolated from carrageen and Salmonella enterica serotype Typhi is associated with TLR4. There are three types of galactan glycosidic bonds: α-(1,3), α-(1,4), and β-(1,4) glycosidic bonds (Table 4). Additionally, some α-(1,3), α-(1,4), and β-(1,4) glycosidic linkage polysaccharides are associated with TLR4. 4.2.1. α-(1,3) and β-(1,4)-galactose polymers Carrageenan: Carrageenan is widely used as a food additive in the western diet. The chemical structure of carrageen consists of sulfated galactose residues that are connected in α-1,3 and β-1,4 linkages. The sites and extent of sulfations and secondary branching may vary among the λ, κ, and ιιιι types of carrageen, which are all derived from red seaweed. All carrageen types contain an unusual α-1,3-galactosidic linkage, which is recognized in humans by anti-Gal antibodies. The molecular mass of λ-carrageen is approximately 1,000 kDa. Experiments with fluorescence-tagged carrageen have shown that exposure to a TLR4 blocking antibody (HTA-125) causes a marked reduction in CGN binding to human intestinal epithelial cells and RAW 264.7 mouse macrophages. In 10ScNCr/23 mouse macrophages, which are deficient in TLR4, carrageen cannot bind. Experiments with TLR4 blocking antibodies and small-interfering RNAs have shown 80% reductions in carrageen-induced increases in Bcl10 and IL-8 (Bhattacharyya et al., 2008).

Thus, these results collectively supported that λ-carrageen is a TLR4 ligand (Bhattacharyya et al., 2010; Borthakur, Bhattacharyya, Anbazhagan, Kumar, Dudeja & Tobacman, 2012; Zhang, Tsai, Monie, Hung & Wu, 2010). 4.2.2. α-(1,4)-galacturonic acid polymers SViCPS: Capsular polysaccharides are a major virulence factor in meningococcal infections and form the basis for serogroup designation and protective vaccines (Ubale, D'Souza, Infield, McCarty & Zughaier, 2013). Capsular polysaccharides, which are expressed by gram-positive bacteria, are covalently linked to a thick, underlying cell wall peptidoglycan to which a number of proteins are also covalently attached (Deng, Kasper, Krick & Wessels, 2000; Jedrzejas, 2004). Capsular polysaccharides expressed by gramnegative bacteria, which have a thin peptidoglycan cell wall, are attached through a labile covalent linkage to the acyl glycerol moiety of the outer membrane. The outer membrane is known to have multiple immunomodulatory properties, in part due to the presence of LPS (Ellis & Kuehn, 2010; Massari, Henneke, Ho, Latz, Golenbock & Wetzler, 2002). The Vi capsular antigen is a α-1,4(2-deoxy)-2-N-acetylgalacturonic acid linear polymer that is variably O-acetylated at the C3 position and is expressed by Salmonella enterica serotype Typhi (SViCPS); however, this antigen is not expressed in nearly all other Salmonella serotypes (Raffatellu, Chessa, Wilson, Dusold, Rubino & Baumler, 2005). Studies have shown that infection with Vi-positive Salmonella typhimurium (C5.507 Vi(+)) or C5.507 Vi(+) results in a significant reduction in cellular trafficking of innate immune cells, including peripheral mononuclear (PMN) and NK cells, as compared to that in SGB1 Vi(-) infected animals. C5.507 Vi(+) infection reduces the number of TNF-α-, MIP-2-, and perforinproducing cells compared with SGB1 Vi(-) infection. The modulatory effects associated with Vi were not observed in MyD88-/- mice and were reduced in TLR4-/- mice (Jansen et al., 2011). 4.3. Mannan Mannan: Mannan is a highly branched polymer. Research has shown that mannan is present in the yeast cell wall with α-(1,6)-mannose as the backbone chain and that most α-(1,2) or α-(1,3) residues are connected with 2–5 mannose side chain residues. Many

higher plants have α-(1,4) mannose as the backbone chain. Using dry konjac powder as a raw material, Japanese researchers successfully prepared powdered mannan. Mannan can also be extracted from yeast and yams (Liu et al., 2008a; Sheng et al., 2006). Mannan isolated from yeast binds to C-type lectins of the mannose receptor family, expressed by APCs, including DCs and macrophages. Because these receptors mediate endocytosis, they have been targeted with ligands to deliver antigens into APCs to initiate immune responses. Immunization with the tumor antigen mucin 1 (MUC1) conjugated to oxidized mannan (OM) or reduced mannan (RM) induces differential immune responses in mice, and only mice immunized with OM-MUC1 exhibit strong MUC1-specific cytotoxic Tlymphocyte responses, which protects them from a MUC1 challenge. Mannan, OM, and RM are capable of stimulating mouse BMDCs in vitro, eliciting enhanced allogeneic T-cell proliferation and enhancing OTI/OTII peptide-specific T-cell responses Experiments have demonstrated that activation of DCs is dependent on TLR4 (Sheng et al., 2006). Moreover, the yeast form of Candida albicans and its mannan and cell wall fractions exhibit higher TNF-α production than the respective preparations from the hyphal form. Only slight TNFα production was induced by the Saccharomyces cerevisiae glucan. Additionally, TNF-α production triggered by reference LPS and purified fungal mannans requires the presence of LPS-binding protein (LBP), and these responses are inhibited by anti-CD14 and antiTLR4 antibodies, but not by anti-TLR2 antibodies. In contrast to the activity of LPS, the activity of purified Saccharomyces cerevisiae mannan is not inhibited by polymyxin B. These findings suggest that the mannan-LBP complex is recognized by CD14 on monocytes and that signaling through TLR4 leads to the production of pro-inflammatory cytokines in a manner similar to that induced by LPS (Tada et al., 2002). 4.4. Fructan PG: There are three fructan glycosidic bond types: β-(2,1), β-(1,6), and β-(2,6) glycosidic bonds (Yoon et al., 2003; Yoon, Kang, Han, Park, Lee & Kim, 2004) (Table 4). Only β-(2,1)-fructan has been reported to have activity related to TLR4. Aqueous extracts of the PG radix contain an inulin-type polyfructose with a (2,1)-linked D-fructose. Water extracts of the roots of PG radix have been found to produce a polysaccharide that contains

an inulin type (2,1) connection in a fructose-based polymer. PG induces NO production by macrophages isolated from C3H/HeN mice, but not by macrophages isolated from C3H/HeJ mice. Moreover, monoclonal anti-TLR4 antibodies block PG-mediated induction of NO production. In addition, LBP and sCD14 have also been found to be involved in the activation of NO production by PG. PG causes degradation of IκB and activation of DNA binding by NF-kB, a downstream transcriptional regulator of TLR4. Taken together, these results suggest that PG-mediated induction of NO production and inducible nitric oxide synthase (iNOS) mRNA expression in macrophages is mediated, at least in part, by the TLR4/NF-κB signaling pathway. Strikingly, short-chain-enriched β-(2,1) fructans promote the balance of regulatory cytokines compared with long chain enriched β-(2,1)-fructans, as measured by IL-10/IL-12 ratios. Activation of reporter cells has shown that signaling is highly dependent on TLRs and their adapter, MyD88. In human embryonic kidney reporter cells, TLR2 is prominently activated, while TLR4, TLR5, TLR7, TLR8, and nucleotidebinding oligomerization domain containing 2 (NOD2) are only mildly activated. The results indicated that β-(2,1)-fructans exert direct signaling effects on human immune cells. By activating primarily TLR2, and to a lesser extent TLR4, TLR5, TLR7, TLR8, and NOD2, β(2,1)-fructan stimulation results in NF-κB/AP-1 activation. The chain length of β-(2,1)fructans is important for the induced activation pattern and IL-10/IL-12 ratios (Vogt et al., 2013). 4.5. Polysialic acid PolySia: Neisseria meningitidis is an encapsulated gram-negative bacterium and causes epidemic bacterial meningitis and fulminant sepsis. Meningococcal strains are classified into serogroups based on the structural differences of their capsules. The Neisseria meningitidis serogroup B capsular polysaccharides (CPS) is composed entirely of sialic acid having an α-(2,8) linkage (Liu, Gotschlich, Dunne & Jonssen, 1971; Tzeng, Datta, Strole, Lobritz, Carlson & Stephens, 2005) (Table 4). CPS is the major virulence factor in infections caused by Neisseria meningitidis and forms the basis for meningococcal serogroup designation and protective meningococcal vaccines. CPS polymers are anchored in the meningococcal outer membrane through a 1,2-diacylglycerol moiety. Well-

established human and murine macrophage cell lines and TLR-stably transfected HEK cells have been stimulated with CPS purified from an endotoxin-deficient meningococcal serogroup B NMB-lpxA mutant, and CPS was shown to induce inflammatory responses via TLR2 and TLR4-MD-2. Moreover, CPS induces IL-8 release from HEK cells stably transfected with TLR2/6, TLR2, TLR2/CD14, and TLR4/MD-2/CD14, but not untransfected HEK cells. A monoclonal antibody targeting TLR2 but not an isotype control antibody blocked CPS-induced IL-8 release from TLR2/TLR6-transfected HEK cells. Additionally, significant reductions in TNF-α and IL-8 release were observed when TLR4/MD-2-CD14transfected THP-1 and HEK cells , but not TLR2- or TLR2/TLR6-transfected HEK cells, were stimulated with CPS in the presence of the lipid A antagonist and MD-2 binding partner eritoran (E5564). Similar reductions in NO and TNF-α release were observed in RAW 264.7 cells in the presence of eritoran. Thus, these data suggest that innate immune recognition of meningococcal CPS by macrophages can occur via TLR2 and TLR4-MD-2 pathways (Zughaier, 2011).

Table 4 TLR4-related homoglycan glycosidic bond types.

Homoglycan Glucan

Galactan

Glycosidic

TLR4

bond types

correlation

α-(1,4)

Activation

α-(1,6)

Activation

β-(1,3)

Activation

(Harnack, Eckert, Fichtner & Pecher,

β-(1,4)

Activation

2010) (Li et al., 2004)

β-(1,6)

Not reported

α-(1,3)

Activation

(Bhattacharyya et al., 2008;

α-(1,4)

Inhibition

Bhattacharyya et al., 2010) (Raffatellu, Chessa, Wilson, Dusold,

β-(1,4)

Activation

Rubino & Baumler, 2005) (Mikshina, Gurjanov, Mukhitova,

β-(2,1)

Activation

β-(1,6)

Not reported

Rasmussen, 2012)

β-(2,6)

Not reported

(Szwengiel, Czarnecka, Gruchala &

β-(1,2)

Not reported

Czarnecki, 2009) (Dang, Johnson & Bundle, 2012; Wu,

β-(1,3)

Not reported

Lipinski, Paszkiewicz & Bundle, 2008) (Cai, Wu & Crich, 2009)

β-(1,4)

Not reported

(Mizutani et al., 2012)

α-(2,8)

Activation

(Nagae et al., 2013)

α-(2,9)

Not reported

(Freiberger et al., 2007)

Reference

(Smiderle et al., 2011)

(Smiderle et al., 2011)

Petrova, Shashkov & Gorshkova, Fructan

Mannan

Polysialic

(Harrison, Xue, Lane, Villas-Boas &

acid

5. Molecular weights Current research has proven that polysaccharide activity is closely related to MW. Polysaccharides that are too small or too large often do not have activity, and a suitable molecular polymerization degree will have a biological regulatory response. Interestingly, the MW range of reported TLR4 polysaccharide ligands is widely distributed from 5 kDa (apple polysaccharides) to 2,400 kDa (the Phoma herbarum polysaccharide YCP). However, most of the MWs of polysaccharide ligands are between 10 and 1,000 kDa (Table 5), suggesting that these polysaccharides may be much more active. However, it is still unclear whether there are differences in the binding affinities of different polysaccharides with TLR4 and whether such differences in binding affinity may be dependent on MW. We believe that the TLR4/MD-2 complex structure itself makes it difficult to affinity bind with small-molecular-weight oligosaccharides (see section 8.1). Interestingly, hyaluronic acid oligosaccharide activity is reported to be associated with TLR4 (Termeer et al., 2002). We speculated that oligomeric hyaluronic acid activity may occur through direct interaction with TLR4/MD-2, but not the TLR4/MD-2 dimer, thereby activating relevant signaling pathways.

Table 5 Molecular weights (MW s) of TLR4-related polysaccharides. Genus

Polysaccharides

MW (kDa)

Reference

Malus

APs

5–10

(Zhang et al., 2012b)

Angelica gigas Nakai

Angelan

10

(Kim et al., 2007)

Platycodon grandiflorum

PG

> 8.9

(Yoon et al., 2003)

Astragalus mongholicus

APS

20–60

Carthamus tinctorius L.

SF1/SF2

> 100

(Liu, Yao, Yu, Dong & Sheng, 2011a) (Ando et al., 2002)

Lycium barbarum L.

LBPF4-OL

181

Tinospora cordifolia

G1-4A

2200

Coriolus versicolor

Krestin

94

Polyporus umbellatus

PPS

160

Sparassis crispa

Sparan

510

Cordyceps militaris

Cordlan

576

(Kim et al., 2010b; Park, Shim, Choi & Park, 2009) (Kim et al., 2010a)

Phoma herbarum

YCP

2400

(Zhu et al., 2014)

Acetobacter xylinum

AC-1

508

(Li et al., 2004)

Rhodophyta

Carrageenan

1,000

Chlorella pyrenoidosa

CWSP

> 1,000

(Bhattacharyya et al., 2008; Borthakur, Bhattacharyya, Anbazhagan, Kumar, Dudeja & Tobacman, 2012) (Hsu, Jeyashoke, Yeh, Song, Hua & Chao, 2010b)

(Zhang et al., 2014a; Zhang et al., 2014b; Zhang et al., 2011) (Raghu, Sharma, Ramakrishnan, Khanam, Chintalwar & Sainis, 2009) (Tsukagoshi, Hashimoto, Fujii, Kobayashi, Nomoto & Orita, 1984) (Li, Xu & Chen, 2010)

Fig. 2. Molecular weights of TLR4-related polysaccharides. ● represents 10–200-kDa polysaccharides, Ο represents 1200–1000-kDa polysaccharides, and Ο represents 11000–2400-kDa polysaccharides.

6. Immune target cells A recent study found that TLR4 is widely distributed on different cell types, and all immune system cells express TLR4, including monocyte/macrophages, DCs, B lymphocytes, T lymphocytes, γδ T cells, granulocytes, NK cells, and natural killer T (NKT) cells (Cui, Kang, Cui & He, 2009; Sabroe, Jones, Usher, Whyte & Dower, 2002). However, only some target cells have functions associated with TLR4 (Table 6). Additionally, all these cells are likely to be targets of TLR4-related polysaccharides. Lycium barbarum polysaccharide, rhamnogalacturonan II, and angelan can induce DC maturation and activation and strengthen the antigen-presenting ability of DCs (Kim et al., 2007; Park et al., 2013; Zhu, Zhang, Shen, Zhou & Yu, 2013b). Safflower polysaccharide and Acanthopanax senticosus polysaccharide have been reported to activate macrophages and stimulate macrophage phagocytosis and antigen-presenting function (Ando et al., 2002; Han et al., 2003). G1-4A and Reishi can activate B lymphocytes and promote the release of immunoglobulin from plasma cells (Lin et al., 2006; Raghu, Sharma, Ramakrishnan, Khanam, Chintalwar & Sainis, 2009). Yamoa polysaccharide and Astragalus polysaccharides can prime T cells (Graff et al., 2009), adjust the balance of T cell subgroups, and affect the function of regulatory T cells (Tregs) (Feng et al., 2013). These results support the use of polysaccharides as vaccine adjuvants. Some other polysaccharides, such as Strongylocentrotus nudus egg polysaccharide, can promote NKmediated cytotoxicity in the NK1.1 cell population and IL-2 and IFN-γ secretion, which explains the anticancer activity of this polysaccharide (Ke et al., 2014). Additionally, polysaccharides have a significant protective effect on the nervous system by inhibiting the central inflammatory response and regulating immune reactions (Cui, Jia, Zhang, Zhang & Wang, 2012; Kang et al., 2012; Teng, Li, Cheng, Zhou, Shen & Wang, 2013; Yao, Xu & Wu, 2014). Moreover, polysaccharides can reduce the negative effects of alcohol on liver structure and function and promote protective effects against oxidative injury in hepatocytes (Ozalp et al., 2014; Wang, Dong, Ma, Cui & Tong, 2014). Notably, polysaccharide-protein interactions are weaker than protein-protein interactions. Several mechanisms can enhance polysaccharide-protein interactions, including higher-order

protein recognition, multisite interactions, and repeated binding (Nagae & Yamaguchi, 2014). Additionally, the polysaccharide-induced increase in TLR4 expression in target cells may enhance TLR4-polysaccharide interactions.

Table 6 Targeting of immune system cells by TLR4-related polysaccharides. Immune target cells

Polysaccharides

Effects on target cell

Reference

Dendritic cells

AAP, Angelan, RG-II, Cordlan, PPS, PSK, Sparan, LNFPIIIDex

↑maturation, Activation, ↑antigenpresenting

(Kim et al., 2011; Zhu, Zhang, Shen, Zhou & Yu, 2013a)

Macrophages

AAP, SF1/SF2, APS, G1-4A, LBPF4-OL, ZPF1, Inulin, PG, GXM, PPS, PSK, Mannan, YCP, XG, PolySia, AC-1, CWSP, CAF, Carrageenan

Activation ↑ phagocytosis, ↑ cytokines

(Han et al., 2003)

B cells

APS, G1-4A Inulin, PG Reishi-F3 YCP

Activation, ↑immunoglobulin

(Raghu, Sharma, Ramakrishnan , Khanam, Chintalwar & Sainis, 2009)

αβ T cells

APS Inulin PSK Xanthan gum (XG)

Activation, ↑ proliferation ↑ cytokines

(Liu, Yao, Yu, Dong & Sheng, 2011a)

NK cells

Inulin, XG

↑ Anti-tumor activity

(Ke et al., 2014)

NKT cells

LPS

Activation, ↑ IFN-γ, ↓ IL-4

(Kim, Kim, Kim, Oh & Chung, 2012)

↑ increase; ↓ decrease; NK cells, natural killer cells; NKT cells, natural killer t-cells. （For color reproduction on the Web and in print）

7. Primary structure and activity relationships TLR4 is a pattern recognition receptor (PRR) that belongs to the type I transmembrane protein family and is localized at the cell surface. The extracellular leucine-rich repeat (LRR) domain can recognize biological macromolecules. TLR4 ligands can be divided into four categories: (1) polysaccharides, first shown to bind with TLR4 by Japanese scholars and confirmed by Chinese and South Chesapeake scholars (Han et al., 2003); (2) glycolipids, including G(-) bacterial LPS and lipid A; (3) DNA, specifically CpG motifs of bacterial DNA; and (4) protein, including EDA fibronectin, fibrinogen, and tenascin-C (Jia, Niu, Cong, Zhang & Zhao, 2014). 7.1. Monosaccharide types and activity relationships The collected data in this review show that monosaccharide composition is a critical factor that determines polysaccharide activity through TLR4. TLR4-related activity occurs with polysaccharides that are widely distributed throughout the world, both in prokaryotes and eukaryotes. Additionally, the monosaccharide compositions of polysaccharides from higher plants are highly complex, whereas those from animal sources may be the most simple; the monosaccharide compositions of polysaccharides from bacteria, fungi, and algae are of moderate complexity between those from plants and animals. We also found that glucose, galactose, and mannose are the most frequent monosaccharides present in polysaccharides. Additionally, TLR4-related homoglycans contain glucan, galactan, and mannan, which are known TLR4 ligands. Furthermore, these three monosaccharide types, along with other monosaccharide combinations, produce polysaccharides with high TLR4related activities. Thus, these three monosaccharides are the pharmacophore of TLR4related active polysaccharides (Fig. 3). It is worth pointing out that Inulin-type fructans and outside source polysialic acid capsular polysaccharides are to have reported activity that is relate to TLR4 signalling (Vogt et al., 2013). However, at present, only two fructan types, one of which is Levan, a major fraction of fermented soybean mucilage, and the other being a polysaccharide that is isolated from the Platycodon grandiflorum radix, were reported to have TLR4-dependent activity (Yoon et al., 2003). Polysialic acid is mainly distributed in vertebrate cell

gangliosides and glycoproteins and in some microorganisms, such as Escherichia coli K235. However, compared with glucan polysaccharides, the acquired access of polysialic acid is still limited. Additionally, polysialic acid is a linear polysaccharide with glycoside bonds only at α-(2,8) and α-(2,9). This may explain why interactions between polysialic acid and TLR4 have rarely been reported. Additionally, there are many other homoglycans, such as xylan, araban, and rhamnosan, that have not been reported to be associated with TLR4 signaling. Of these homoglycan, it is unknown whether xylan is related to TLR4 signaling. Additionally, arabinose and rhamnose are rarely found in nature in the homoglycan form; however, in the heteroglycan form, such as arabinogalactan and rhamnogalacturonan, these hetero-polysaccharides have been reported to have TLR4related signaling activity. 7.2. Glucosidic bonds and activity relationships The reported TLR4-related homoglycans glycosidic bond types are listed in Table 4. Glucans are found in the α and β configurations with four glycosidic bond types: β-(1,3) (Hida, Ishibashi, Miura, Adachi, Shirasu & Ohno, 2009), β-(1,4), α-(1,4), and β-(1,6) (Camelini, Maraschin, de Mendonca, Zucco, Ferreira & Tavares, 2005). The activities of the β-(1,3)-, β-(1,4)-, and α-(1,4)-linked glucan backbones have been reported to be related to TLR4 signaling. However, no reports have described the relationship between β-(1,6)-linked glucan backbones and TLR4. The β-(1,6)-linked glucan backbone has been reported to be associated with dectin-1. A research study found that the complex of agaricus-derived β-(1,6)-glucans and laminarin, but not dextran and islandican, directly bound to dectin-1 (Yamanaka et al., 2012). In galactan, the α-(1,3), α-(1,4), and β-(1,4) glucosidic bonds are all correlated with TLR4 signaling, indicating that galactose itself may be a type of monosaccharide recognition model for TLR4. The mannan from Saccharomyces cerevisiae is also related to TLR4 signaling; however, the glucosidic bond structure involved in this interaction is still unknown. There are three types of fructan glycosidic bonds: β-(2,1), β-(1,6), and β-(2,6) glycosidic bonds. Only the β-(2,1)-fructan bond has been reported to have TLR4-related activity. Additionally, α-(2,8)- and α-(2,9)linked sialic acids have been isolated; however, only α-(2,8) polysialic acid was shown to

be related to TLR4 activity. Thus, only specific homoglycan linkages are related to TLR4 signaling. 7.3. MW and activity relationships Polysaccharide MW distributions greatly affect their biological activities (Guo et al., 2008; Nishino, Aizu & Nagumo, 1991). Many studies have shown that anticoagulation and antioxidant activities are characteristic of low-MW polysaccharides (Fu et al., 2014; Ke, Wang, Sun, Qiao, Ye & Zeng, 2013; Sheng & Sun, 2014; Tian, Zha, Pan & Luo, 2013; Wang, Zhang, Yao, Zhao & Qi, 2013; Zhang, Wang, Zhao & Qi, 2014). However, the immunostimulatory activities of polysaccharides are obviously different, whereby both oligosaccharides and high-MW polysaccharides have immunostimulatory activities. For example, hyaluronic acid oligosaccharides have been reported to suppress TLR3dependent cytokine expression in a TLR4-dependent manner (Kim, Muto & Gallo, 2013). The water-soluble polysaccharide SCPP11 (3.4 kDa) can increase thymus indexes and IL2 and TNF-α levels in serum in vivo and significantly enhance phagocytosis and NO production by RAW264.7 cells in vitro (Zhao et al., 2013). North American GS polysaccharide (> 100 kDa) extracts likely trigger the MAPK (ERK-1/2), phosphoinositol 3kinase (PI3K), p38, and NF-κB signaling pathways in peripheral blood mononuclear cells (PBMCs), resulting in the induction of a Th1 transcriptional profile (Lemmon, Sham, Chau & Madrenas, 2012). Additionally, hyaluronic acids with different MW s, including LMWHA-1 (145 kDa), LMWHA-2 (45 kDa), and HA (1050 kDa), promote splenocyte proliferation, increase acid phosphatase activity in peritoneal macrophages, and improve the capacity for peritoneal macrophages to take up neutral red in vitro in a concentration-dependent manner. Furthermore, LMWHA-1 and LMWHA-2 exhibit much stronger immunostimulatory activity than HA (Ke, Wang, Sun, Qiao, Ye & Zeng, 2013). The above results indicate that polysaccharides with a wide range of MW s can induce immune reactions. Notably, chitin is a size-dependent regulator of innate immunity. Researchers have found that large chitin fragments are inert, whereas both intermediate- (40–70 μm) and small-sized chitin (SC; < 40 μm, largely 2–10 μm) stimulate TNF production. In contrast, only SC induces IL-10

production (Da Silva, Chalouni, Williams, Hartl, Lee & Elias, 2009). Thus, the anti- or proinflammatory activities of chitin are dependent on MW . No studies have reported variations in the TLR4 agonist activities of low- and high-MW polysaccharides. However, many TLR4-related polysaccharide ligands have been identified, with varying MW s (Table 5). The TLR4-related polysaccharide with the highest MW is YCP from Phoma herbarum, with a mean MW of 2,400 kDa, and that with the lowest MW is the polysaccharide from apples, with a mean MW of 5–10 kDa. Therefore, the MW range of TLR4 polysaccharide ligands is consistent with that of immunostimulatory polysaccharides. As shown in Table 6, polysaccharides with MW s of less than 1,000 kDa are present in a 13/15 ratio, whereas those with MW s of over 200 kDa are present in a ratio of 8/15 (Fig 3). These data indicate that polysaccharides with MW s ranging from 10 to 1000 kDa are in a suitable for TLR4 activation, particularly those with MW s of 10–200 kDa, which are the most active.

Fig. 3. Monosaccharide compositions and frequency analysis of TLR4-related polysaccharides. 8. Polysaccharides and protein interaction models The three-dimensional (3D) TLR4/MD-2 structure and LPS-TLR4 interaction model have been reported. The extracellular domains of TLR4 consist of LRRs with a horseshoelike shape (Kim et al., 2007a). In the inner surface of the horseshoe-shaped molecules are β sheets, which contain conservative LRRs. Additionally, an α-helix structure is present in the outer surface. In this structure, the conserved leucine residues form a hydrophobic core when combined with the ligand, and the other nonconserved amino acid residues are exposed in the peripheral region of the horseshoe-shaped molecules. LRR domains are involved in protein-protein interactions, most of which participate in signal transduction pathways (Groves & Barford, 1999). The TLR4/MD-2-LPS interaction model showed that TLR4/MD-2 complex dimerization is vital for TLR4 signal activation. TLR4 adopts the characteristic horseshoe-like shape of the LRR superfamily. MD-2 has a β-cup fold structure that is composed of two antiparallel sheets that form a large hydrophobic pocket for ligand binding. The main dimerization interface of MD-2 is located on the opposite side of the primary interface and interacts with LRR modules 15–17 in the C-terminal domain of TLR4. LPS binds to this pocket and directly mediates dimerization of the two TLR4/MD-2

complexes. Additionally, in this model, the lipid A portion has been identified as the active center, which is responsible for LPS-induced macrophage activation. Five carbon chains of lipids are completely buried inside this pocket, and one chain is partially exposed to the MD-2 surface; these lipids make up the core hydrophobic interface for interaction with TLR4. The total number of lipid chains is the most important factor mediating this interaction. Lipid A has six lipid chains and exhibits optimal inflammatory activity; however, five of these lipid chains in lipid A are 100-fold less active than the sixth chain. Furthermore, those with four lipid chains, such as eritoran, completely lack agonistic activity (Rossignol & Lynn, 2005; Teghanemt, Zhang, Levis, Weiss & Gioannini, 2005). Interestingly, Salmonella lipid A is inactive in human macrophages, despite the high activity of Salmonella LPS. In THP-1 human monocytic cells, Salmonella lipid A and synthetic Salmonella-type lipid A (516) do not induce NF-κB-dependent reporter activity up to 1 μg/mL, whereas strong activation is observed in response to Salmonella LPS. These results indicate that the polysaccharide portion that is covalently bound to lipid A plays a predominant role in Salmonella LPS-induced activation of NF-κB through human CD14/TLR4/MD-2 (Muroi & Tanamoto, 2002). Finally, this model also showed that two LPS phosphate groups are involved in polymer formation, which is critical for TLR4 activation. The two phosphate groups of lipid A bind to the TLR4/MD-2 complex by interacting with positively charged residues in TLR4/MD-2, facilitating the formation of a hydrogen bond with MD-2. Thus, the formation of poly-TLR4/MD-2 complexes is the key to activating the TLR4 signaling pathway. Polysaccharide-protein binding affinities are significantly weaker than protein-protein interactions; therefore, the appropriate enhancement mechanism is important. Lectins that bind to linear polysaccharides are classified as either exo- or endo-types. Exo-type lectins interact with the terminal units of polysaccharides and bind glycan chains in partially sealed clefts. Lectins of this type specifically bind to the terminal cap structure. In contrast, endotype lectins interact with internal polysaccharide units using open clefts. Endo-type lectins enhance binding affinity to polysaccharide ligands by three mechanisms: (1) multisite interactions, which occur when a long polysaccharide binds two or more lectins

simultaneously; (2) repeated binding, in which one lectin repeatedly dissociates and rebinds to or slides on the polysaccharide or slowing of protein dissociation may enhance the apparent affinity, which requires that the protein has ligand-binding pockets open at both ends; and (3) recognition of higher-ordered polysaccharide structures, called the conformational epitope hypothesis. This theory remains speculative; however, in this type of interaction, a polysaccharide of a certain chain length forms a higher-ordered conformation, such as a helix, which assists in protein recognition and tighter binding. The chain length at which the polysaccharide forms a higher-ordered structure depends on the glycosidic linkage and sugar type (Nagae & Yamaguchi, 2014). Tighter binding of endotype proteins to longer polysaccharides is often achieved by combining all three mechanisms (Nakata & Troy, 2005; Sato & Kitajima, 2013). In short, the enhancement mechanism is very important in the polysaccharides-protein interaction. The crystal structure of the single-chain variable fragment of mAb735 (scFv735) in complex with octasialic acid has been reported. In this structure, scFv735 was found to interact with ligands by an endo-type mechanism, and several scFv735 molecules were shown to bind simultaneously to the long polysialic acid chain. Multiple molecules could bind to the linear polysialic acid chain, resulting in high affinity for polysialic acid; however, the increase in affinity likely results from the decreased off-rate of the protein. Moreover, although the functions of polysialic acid may be closely related to their 3D structures, the conformation of polysialic acid is still unclear (Brisson, Baumann, Imberty, Perez & Jennings, 1992; Henderson, Venable & Egan, 2003; Yamasaki & Bacon, 1991; Yongye, Gonzalez-Outeirino, Glushka, Schultheis & Woods, 2008). However, it is clear that higherorder structures may affect polysaccharide binding with proteins, as has been demonstrated with glucan. Although the primary structures of glucans from different sources have been reported to have many similarities, their biological activities are significantly different. The structure-activity relationships of glucans are relatively clear, as was discussed in an outstanding review by Sletmoen and Stokke (2008); higher-order (1,3)-β-D-glucan structures influence biological activities and complexation abilities. Moreover, the different glucan activities may be related to the primary and higher-order

structures of glucans, some of which do not have three-ply helix structures (Levy-Assaraf et al., 2013). Research on the TLR4/MD-2 crystal structure, LPS-TLR4/MD-2 interaction model, linear polysaccharide-lectin interaction model, and the primary structures of the abovementioned 30 TLR4-related polysaccharides has indicated that polysaccharide-TLR4/MD2 interaction models should meet the following five criteria. (1) TLR4/MD-2 complex formation: Polysaccharides must be presented correctly to mediate the formation of poly-TLR4/MD-2 compounds. (2) Monosaccharide type and enhancement mechanism: Glucans, mannans, galactans, fructans, and polysialic acid with reported immunostimulatory activity. In the review, we also found that all polysaccharides can activate/inhibit the TLR4 signaling pathway. Moreover, glucose, mannose, galactose, fucose, and sialic acid may be the key “pharmacophore” of TLR4/MD-2 polysaccharide ligands, and these five monosaccharide types may participate in enhancement of the interaction with TLR4/MD-2. (3) MW : TLR4/MD-2 can bind with both low- and high-MW polysaccharide ligands; thus, this is consistent with the observation that polysaccharides with MWs ranging from 10 to 2,400 kDa can all activate TLR4. (4) Glucosidic bond type of branch or linear polysaccharide: Both branched and linear polysaccharides, including the highly branched β-(1,4) polysaccharide LBPF4-OL from Lycium barbarum and the α-(2,8) linear polysialic acid, can interact with TLR4 and activate the TLR4 signaling pathway. (5) Higher-ordered structure: the model should also be suitable for polysaccharides with higher-ordered structures. Therefore, we propose the following possible polysaccharide-TLR4/MD-2 interaction models (Fig. 4). Low-MW branched polysaccharides may only induce TLR4/MD-2 dimerization (Fig. 4A), and high-MW branched polysaccharides may be able to induce polyTLR4/MD-2 dimerization (Fig. 4B). Obviously, it is difficult for linear polysaccharides to interact with both the TLR4 and MD-2 molecules, and polysaccharides with higher-order

structures may be able to induce TLR4/MD-2 dimerization. Therefore, it is possible that linear polysaccharides may bind with TLR4/MD-2 in the form of a higher-order structure. However, linear polysaccharides with a low MW and higher-order structure may only induce TLR4/MD-2 dimerization (Fig. 4C), and linear polysaccharides with a high MW and higherorder structure may be able to induce poly-TLR4/MD-2 dimerization (Fig. 4D). Although the models can effectively explain the above five features, we believe that a more accurate polysaccharide-TLR4/MD-2 model will have to be validated by structural biology studies.

Fig. 4. Possible TLR4/MD-2 and polysaccharide interaction models. A, Interaction model for a low-molecular-weight branched polysaccharide with a dimerized TLR4/MD-2 complex. B, Interaction model for a high-molecular-weight branched polysaccharide with a TLR4/MD-2 complex polymer. C, Interaction model for a low-molecular-weight linear polysaccharide having a higher-order structure with a TLR4/MD-2 complex dimer. D, Interaction model for a high-molecular-weight linear polysaccharide having a higher-order structure with a TLR4/MD-2 complex polymer. The grey half-arc represents TLR4, the blue points represent polysaccharides, the blue ribbon represents MD-2, and the yellow line represents the cell membrane. （For color reproduction on the Web and in print）

9. Conclusions and outlook In this review, we aimed to present an overview of the primary structures, activity relationships, and possible interaction models of TLR4/MD-2 with polysaccharides. Because many papers have been published on this subject, we were forced to select only the most significant results. Collectively, these studies showed that TLR4-related homoglycan polysaccharides mainly include glucan, galactan, mannan, fructan, and polysialic acid. The monosaccharide composition analysis showed that glucose, galactose, and mannose occur at the highest frequency in all TLR4-related polysaccharides. Moreover, of heteropolysaccharides with TLR4-related activity, glucose (and glucuronic acid), galactose (and galacturonic acid), fructose, and mannose appeared at least once, and most were paired together, suggesting that these monosaccharaides are the key “pharmacophore” of active TLR4-related polysaccharides. In terms of the molecular and glycosidic bond characteristics, TLR4 recognizes an extensive array of polysaccharides with a MW range from 10 to 2,400 kDa; however, polysaccharides with MWs of 10–200 kDa were most often reported. Additionally, active TLR4-related polysaccharides contain mainly α-(1,3), α-(1,4), β-(1,3), and β-(1,4) glycosidic bonds. TLR4 expression was found in all immune cell types, although only some immune cells are recognized as target cells for TLR4-related polysaccharides (e.g., DCs and macrophages). The ability of TLR4related polysaccharides to target other native immune cells, such as γδ T and B-1 cells, is still unclear. Finally, only branched polysaccharides or linear polysaccharides with a higher-order possess special structure properties that allow for binding with the TLR4/MD2 complex. Future studies are needed to determine the structures of high-order galactan, mannan, fructan, and poly sialic acid and to elucidate efficient methods for separation and purification of polysaccharides. Additionally, investigation of the structure-activity relationships of other polysaccharides and receptors, such as lectins, is also needed.

Acknowledgments This work is supported by National Natural Science Foundation of China (No. 81102451).

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Capsular