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Glycobiology vol. 22 no. 1 pp. 146–159, 2012 doi:10.1093/glycob/cwr122 Advance Access publication on August 26, 2011 Essential role of Toll-like receptor 2 in macrophage activation by glycogen Ryo Kakutani1,2, Yoshiyuki Adachi3, Hiroki Takata2, Takashi Kuriki2, and Naohito Ohno3 2 Institute of Health Sciences, Ezaki Glico Co., Ltd., 4-6-5, Utajima, Nishiyodogawa-ku, Osaka 555-8502, Japan and 3Laboratory for Immunopharmacology of Microbial Products, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo 192-0392, Japan Received on April 30, 2011; revised on August 22, 2011; accepted on August 23, 2011 We prepared enzymatically synthesized glycogen (ESG) with the same characteristics as natural glycogen and investigated whether the macrophage-stimulating activity of glycogen was related to Toll-like receptors (TLRs), which are important receptors for innate immunity. ESG induced no nuclear factor-kappa B (NF-κB) activity in TLR4/MD-2/ CD14-expressed human embryonic kidney 293 (HEK293) reporter cells, whereas this polysaccharide did activate peritoneal exude cells (PECs) derived from TLR4-deficient mice at the same level as those from wild-type (WT) mice. Similarly, ESG did not activate HEK293 cells expressing TLR3, 5, 7, 8 or 9, suggesting that these TLRs were irrelevant to the activity of ESG. In contrast, ESG enhanced the NF-κB activity of TLR2-expressed HEK293 reporter cells in a concentration-dependent manner. Furthermore, the cell-stimulating activity of ESG was remarkably lower for PECs from TLR2-deficient mice compared with those from WT mice. The activity of ESG completely disappeared after treatment with a glycogen-degrading enzyme, indicating that the activity derived from ESG itself and not from contamination with canonical TLR2 ligands such as bacterial lipopeptides. Moreover, it was clarified by ELISA that ESG was directly bound to TLR2. Taken together, these results demonstrated that TLR2 directly recognizes glycogen and that the recognition activates immunocytes such as macrophages to enhance the production of nitric oxide and inflammatory cytokines. In addition, it was suggested that TLR2 could be involved in the glycogen activity in vivo. We propose that glycogen act as an activator to potentiate the host defense through TLR2 on the macrophage. Keywords: carbohydrate / glycogen / immunostimulating activity / macrophage / Toll-like receptor 2 1 To whom correspondence should be addressed: Tel: +81-6-64778425; Fax: +81-6-64778362; e-mail: [email protected] Introduction Glycogen is a highly α-1,6-branched α-1,4 glucan with a high molecular weight ranging from 106 to 109 and is found widely in nature. The largest reserves of glycogen in a mammal are found in the liver and skeletal muscle, whereas a small quantity of glycogen is also present in other tissues such as brain, thymus, heart, skin, placenta and leukocytes (Scott and Still 1968; Harmon and Phizackerley 1983; Blows et al. 1988; Salmoral et al. 1990; Brown 2004; Taegtmeyer 2004). It is also well recognized that the major functions of glycogen are to supply energy in the muscle and to release glucose to the bloodstream from the liver (Geddes 1986). However, some reports have suggested that the intracellular glycogens in the liver and muscle play additional roles beyond its simple function as an energy depot (Greenberg et al. 2006). In contrast to the liver and muscle glycogens, studies of possible roles of the polysaccharide in other tissues have only been limited. Recently, it has been reported that glycogens extracted from scallops and oysters possess immunomodulating activities (Takaya et al. 1998). However, the same report revealed that other preparations of glycogen have little or no activity. The authors suggested that the difference in activities might result from differences in the fine structures of glycogen preparations, which could depend on many factors such as extraction methods or source of glycogen. Furthermore, there would be difficulties in accurately determining the immunomodulating activities of glycogens from natural sources due to the high level of contaminants such as lipopolysaccharides (LPSs). To avoid the problems accompanying the preparation of natural glycogen samples, we have prepared enzymatically synthesized glycogen (ESG) as reported previously (Kajiura et al. 2008). It was shown that ESG was the same as naturalsource glycogens in terms of various parameters such as molecular shape and size, molecular weight, branch frequency and chain length (Kajiura et al. 2008, 2010). By using ESG, we revealed that glycogen activated RAW264.7, murine macrophage cell line, to induce the production of nitric oxide (NO) and inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) and that the molecular weight of glycogen was strongly related to its macrophage-stimulating activity (Kakutani et al. 2007, 2008). These results suggested that glycogen may function not only to adjust the blood sugar level but also as an immunomodulating activator. Over the past decade, a large number of studies have revealed that specific innate immune receptors, such as © The Author 2011. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: [email protected] 146 TLR2 plays an important role in the recognition of glycogen Toll-like receptors (TLRs), NOD-like receptors, RIG-I-like receptors and C-type lectin receptors (CLRs), play important roles in host defense (Chamaillard et al. 2003; Takeda et al. 2003; Adachi et al. 2004; Saijo et al. 2007; Yoneyama and Fujita 2007). Especially, the TLRs have been extensively studied and demonstrated to participate in host defense against bacteria, viruses, fungi and parasites (Aliprantis et al. 1999; Takeuchi et al. 1999; Campos et al. 2001). TLRs mediate the recognition of a large array of molecules present in pathogens, triggering the production of pro-inflammatory cytokines, activation of microbicidal mechanisms and the induction of adaptive immunity (Schwandner et al. 1999; Thoma-Uszynski et al. 2001; Iwasaki and Medzhitov 2004). These receptors are single-pass type I transmembrane receptors containing extracellular leucine-rich repeat domains and an intracellular Toll/IL-1 receptor homology domain. Most TLRs share a common signaling pathway involving an intracellular adaptor protein called myeloid differentiation factor 88 (MyD88), phosphorylation of extracellular signal-regulated kinase (ERK) and p38 and downstream activation of the nuclear factor-kappa B (NF-κB), leading to the expression of TNF-α and macrophage inflammatory protein-2 (MIP-2). Among the TLRs, TLR2 and TLR4 are the best characterized. TLR2 recognizes bacterial lipopeptides, lipoproteins and peptidoglycan, whereas TLR4 recognizes the bacterial LPS. TLRs have also been reported to recognize some endogenous ligands such as hyaluronan, biglycan, heat-shock proteins and nuclear proteins (Beg 2002; Wallin et al. 2002; Schaefer et al. 2005; Scheibner et al. 2006). These findings suggest that a number of endogenous molecules may also be potent activators of the innate immune system. The aim of this study was to clarify the mechanism of glycogen-induced activation of the innate immune system. We demonstrated that TLR2 directly recognizes glycogen and plays an important role in cell activation by glycogen. To our knowledge, this is the first report demonstrating the existence of such a receptor for glycogen. Results The macrophage plays an important role in bioactivity of glycogen First of all, we investigated what kind of immunocyte is involved in the activity of glycogen. Cluster of differentiation (CD) 11b+ and CD11b− cells were separated from BALB/c peritoneal exude cells (PECs) by magnetic-activated cell sorting (MACS) and stimulated with ESG or Pam3CSK4 (TLR2 ligand) in the presence of interferon-γ (IFN-γ). ESG activated PEC and CD11b+ cells and significantly induced the production of NO and IL-6 from these cells in a dose-dependent manner (Figure 1A and B). On the other hand, CD11b− cells treated with ESG hardly produced NO and IL-6. These results indicated that ESG specifically activates CD11b+ cells such as macrophage to produce NO and inflammatory cytokines. Next, the necessity of IFN-γ to glycogen activity was examined. CD11b+ and CD11b− cells derived from PECs were cultured with ESG or Pam3CSK4 in the absence or the presence of IFN-γ, and the concentration of NO and IL-6 in the culture medium were determined. IL-6 production from CD11b+ cells by ESG stimulation was observed both with and without IFN-γ (Figure 1B and D). On the other hand, ESG induced NO production from CD11b+ cells in the presence of IFN-γ, whereas ESG could not induce NO in the absence of IFN-γ (Figure 1A and C). Pam3CSK4 stimulation did not induce NO production without IFN-γ, either. It was shown that IFN-γ is not essential for the cell-stimulating activity of ESG, whereas this cytokine is essential for the production of NO in response to ESG. The cell-activation mechanism of glycogen is different from that of LPS, and TLR4 is irrelevant to the activity of glycogen To investigate whether TLR4 is involved in the cellstimulating mechanism of glycogen, we constructed human embryonic kidney 293 (HEK293) cells expressing TLR4, MD-2 and CD14 with two reporter plasmids [ pNF-κB luciferase ( pNF-κB-Luc) and pRL-TK]. The transfectants were treated with ESG or LPS (TLR4 ligand) for 24 h. NF-κB activation was promoted by LPS stimulation in a dose-dependent manner (Figure 2A). In contrast, NF-κB activation was not observed with ESG stimulation (Figure 2B). Furthermore, the immunostimulating activities of ESG were investigated with PECs derived from TLR4 knock-out (KO) mice. Compared with wild-type (WT) PECs, LPS was not able to activate TLR4KO PECs to produce TNF-α, while TLR4KO PECs stimulated with ESG significantly produced TNF-α at the same level as WT PECs (Figure 2C). Moreover, real-time polymerase chain reaction (PCR) analysis revealed that TNF-α gene expression was significantly increased by ESG or Pam3CSK4 stimulation but not by LPS stimulation in TLR4KO PECs (Figure 2D). These results indicated that TLR4 is irrelevant to the cell-stimulating mechanism of glycogen. TLR2 is responsible for glycogen-induced NF-κB activation and production of NO and inflammatory cytokines by macrophages We examined whether TLR2, another major receptor in the TLR family, is involved in cell activation by glycogen. HEK293 reporter cells expressing TLR2 with two reporter plasmids were treated with several stimuli for 24 h. Pam3CSK4 and ESG promoted NF-κB activation in a dose-dependent manner (Figure 3A and B). In contrast, TLR2 transfectants were not activated by LPS or CpG-ODN (TLR9 ligand) at all (Figure 3C and D). Another study investigated whether ESG activates RAW264.7 cells pre-treated with the TLR2 neutralizing antibody and found that levels of gene expression for TNF-α, IL-6, IL-1β and nitric oxide synthase 2 (NOS2) by ESG stimulation were suppressed with the neutralizing antibody (data not shown). These results suggested that TLR2 is involved in the cell-activation mechanism of glycogen. Glycogen stimulation is strikingly reduced in TLR2KO, TLR2/4-double KO and MyD88KO cells To clarify the possible role of TLR2 in the activity of ESG, we used PECs from TLR2KO mice. Compared with WT PECs, TLR2-deficient PECs were not activated by Pam3CSK4 at all (Figure 4). On the other hand, imiquimod (TLR7 ligand) and 147 R Kakutani et al. Fig. 1. The macrophage-stimulating activity of ESG. (A and B) Participation of the macrophage in ESG activity. CD11b+ and CD11b− cells (1 × 106 per 1 mL) derived from BALB/c PEC were incubated with saline, ESG (50–500 μg/mL) or Pam3CSK4 (TLR2 ligand; 10 ng/mL) in the presence of IFN-γ (10 ng/mL) for 48 h, then NO (A) and IL-6 (B) concentrations in the supernatants were determined. (C and D) Role of IFN-γ in the macrophage activation by ESG. CD11b+ and CD11b− cells (1 × 106 per 1 mL) were cultured with saline, ESG (50–500 μg/mL) or Pam3CSK4 (10 ng/mL) in the absence of IFN-γ for 48 h; thereafter, NO (C) and IL-6 (D) concentrations in the fluid were assayed. Data are represented as the mean ± SD from three independent experiments (each experiment was performed in triplicate). Pam3 in the graph indicates Pam3CSK4. Significant differences from the non-specimen group using one-way ANOVA with Dunnett’s multiple comparisons test are indicated as follows: *P < 0.05 and **P < 0.01. LPS equally activated the WT and TLR2KO PECs to induce the production of NO, IL-6 and MIP-2. Interestingly, the stimulating activity of ESG decreased strikingly with the TLR2KO PEC compared with the WT PEC. Taken together, these findings demonstrated that TLR2 played an important role in the recognition of glycogen to facilitate the production of various inflammatory cytokines by macrophages. In addition, to clarify the cell signaling mechanism of glycogen stimulation, we examined the phosphorylation of several proteins related to NF-κB and the mitogen-activated protein kinase (MAPK) signaling pathway in glycogenactivated cells by western blot analysis. PECs from BALB/c mice were treated with saline, ESG or Pam3CSK4. As a result, ESG enhanced the phosphorylation of several proteins, including ERK1/2, p38, IκB-α, p65 and Akt (Figure 5A). ESG promoted the phosphorylation of those proteins more weakly than Pam3CSK4, but the cell signaling route originating with glycogen stimulation was similar to that of Pam3CSK4 stimulation, except for JNK. It was shown that the stimulation signals from glycogen are transduced via the NF-κB and MAPK signaling pathways as well as that of the TLR2 ligand, and thereafter the stimulating signals facilitate the various innate immune responses of macrophages. We also tested the effect of ESG stimulation on the phosphorylation of MAPK- and NF-κB-related proteins in TLR2KO PEC. Predictably, ESG and Pam3CSK4 stimulation 148 only very weakly enhanced the phosphorylation of ERK, p38 and NF-κB p65 proteins in TLR2-deficient PECs, whereas imiquimod and LPS stimulation activated WT and TLR2KO PECs equally (Figure 5B). These results supported the profiles of cytokine production in Figure 4 and showed that TLR2 plays an important role in the activation of MAPK and NF-κB pathways by glycogen stimulation. Moreover, we examined the activity of ESG on PECs from TLR2/4-double KO (DKO) and MyD88KO. MyD88 is an important signaling adaptor for TLRs and the essential for the cell activation via TLR2. As a result, ESG stimulation produced very little activation in PECs from these KO mice as well as PECs from TLR2KO (Figure 6). These findings did not conflict with the data that TLR2 plays an important role in the activity of ESG at all. TLRs other than TLR2 are not involved in glycogen activity To investigate the relationship between several TLRs and the cell-activation mechanism of glycogen, the effects of ESG on the NF-κB activation of HEK293 cells expressing TLR2, 3, 4, 5, 7, 8 and 9 were tested. Each transfectant was markedly activated by corresponding positive control ligands, while transfectants other than HEK293 expressing TLR2 were not stimulated by ESG (Figure 7). We showed that TLR3, 4, 5, 7, 8 and 9 are not related to the recognition of glycogen. TLR2 plays an important role in the recognition of glycogen Fig. 2. Action of ESG on TLR4. (A and B) The activity of LPS (TLR4 ligand) and ESG for HEK293 cells expressing TLR4/MD-2/CD14. HEK293 cells transfected with reporter plasmids, TLR4, MD-2 and CD14 expression plasmids were stimulated by LPS or ESG at each indicated concentration for 24 h. The stimulation of transfectants by LPS or ESG was carried out in the ranges of 0–200 ng/mL and 0–400 μg/mL, respectively. The relative activity was calculated as the ratio of pNF-κB-Luc (firefly) activity to pRL-TK (Renilla) activity. Data are represented as the mean ± SD from three independent experiments (each experiment was performed in quadruplicate). Significant differences from the non-specimen group using one-way ANOVA with Dunnett’s multiple comparisons test are indicated as follows: *P < 0.05 and **P < 0.01. (C and D) The immunostimulating activity of ESG for TLR4KO mice PEC. PECs (5 × 105 per 1 mL) derived from WT (BALB/ c) or TLR4KO mice were cultured with either saline, ESG (200 μg/mL), LPS (100 ng/mL) or Pam3CSK4 (10 ng/mL) in the presence of IFN-γ (10 ng/mL). (C) After 48 h, culture fluids were withdrawn, and the amount of TNF-α in the fluid was determined with ELISA. (D) After 5 h, total RNA extracted from stimulated PECs was converted to cDNA by using reverse transcriptase, then the amount of TNF-α gene expression in cDNA samples was quantitatively evaluated with real-time PCR. Data are represented as the mean ± SD. ***Significantly different (P < 0.001) between WT and TLR4KO by an unpaired t-test. Digestion of glycogen with glucoamylase abrogated stimulating effects of glycogen on macrophages To prove that the macrophage-stimulating activity of ESG was not due to contamination with TLR2 ligands like bacterial lipopeptides, ESG degradation products were prepared by using glucoamylase and used in the stimulation test of RAW264.7 in the presence of IFN-γ. Glucoamylase is well known as an α-glucan-degrading enzyme that converts glycogen to glucose by degrading both α-1,4 and α-1,6 linkages. Glucoamylase treatment completely abolished the immunostimulating activity of ESG (Figure 8). In contrast, the activities of Pam3CSK4 and FSL-1 (TLR2 ligands) were entirely unaffected by the same treatment. These results elucidated that the macrophage-stimulating activity of ESG depends on ESG itself and not on any contaminants in the ESG solution. On the other hand, LPS and imiquimod were not bound to TLR2 at all. The binding depended not only on the concentration of ESG but also on the concentration of TLR2 (Figure 9B). Furthermore, natural glycogen prepared from oyster was bound to TLR2 as well as ESG, whereas other glucans such as starch (a-1,4/1,6 glucan), dextran (a-1,6 glucan), schizophyllan glucan (SPG; soluble b-1,3/1,6 glucan) and OX-CA ( particle β-1,3/1,6 glucan) were not bound to it at all (Figure 9C). These results demonstrated that glycogen is directly bound to TLR2. In addition, we examined the binding of ESG to dectin-1, β-glucan receptor. β-Glucans such as SPG and OX-CA were bound to dectin-1, whereas ESG and other ligands were not bound to this receptor at all (Figure 9C). It was revealed that the recognition mechanism of glycogen by a macrophage is different from that of β-glucans. Glycogen is bound to TLR2 directly We examined the binding of TLR2 to glycogen with enzymelinked immunosorbent assay (ELISA). Pam3CSK4, LPS, imiquimod and each glucan were used as a positive/negative control for this experiment. As a result, the binding of ESG to TLR2 was elevated in a dose-dependent manner (Figure 9A). Role of TLR2 to glycogen activity in vivo It was examined whether TLR2 is related to the effect of ESG in vivo. WT (BALB/c) and TLR2KO mice were injected intraperitoneally with ESG, Pam3CSK4 or saline. PECs were retrieved from each injected mice on the day after injection and determined the cell number and the rate of CD11b+Gr-1+ 149 R Kakutani et al. Fig. 3. The activity of ESG on TLR2-expressing HEK293 cells. HEK293 cells transfected with TLR2 expression plasmid and reporter plasmids for 24 h were stimulated by either Pam3CSK4 (0–5 ng/mL), ESG (0–400 μg/mL), LPS (0–1000 ng/mL) or CpG-ODN (TLR9 ligand; 0–1000 ng/mL) at each indicated concentration for 24 h. The relative activity was calculated as the ratio of pNF-κB-Luc (firefly) activity to RL-TK (Renilla) activity. Values are the mean ± SD. Data are representative of three independent experiments (each experiment was performed in quadruplicate). **Significantly different (P < 0.01) from non-specimen group using one-way ANOVA with Dunnett’s multiple comparisons test. cell populations. The number of WT PECs injected with ESG tended to increase compared with saline-treated mice (Figure 10A). Furthermore, the rate of CD11b+Gr-1+ cell populations of WT PECs injected with ESG was significantly elevated (Figure 10B and C). Additionally, MIP-2 concentrations in the peritoneal cavity lavages of ESG-treated mice were significantly higher than those of saline-treated mice (Figure 10D). The results obtained by Pam3CSK4 injection were strikingly similar to those by ESG injection. It was suggested that ESG activated PECs of WT mice to induce the production of MIP-2, and the induced MIP-2 facilitated the migration of the CD11b+Gr-1+ cells such as neutrophil into the peritoneal cavity. On the other hand, when TLR2KO mice were used in the same experiments, ESG scarcely induced MIP-2 production from PEC and the migration of CD11b+Gr-1+ cell (Figure 10A–D). Taken together, these findings indicated that TLR2 also could play an important role in the innate immune response to glycogen in vivo. Discussion In this study, we postulated that specific receptor(s) are involved in the cell-stimulating mechanism of glycogen and examined whether the mechanism was related to several 150 TLRs. TLRs are known as important receptors for the induction of various innate immune responses, as they recognize exogenous ligands such as bacteria, viruses, fungi and parasites (Aliprantis et al. 1999; Thoma-Uszynski et al. 2001; Takeda et al. 2003), while these receptors also recognize the endogenous ligands in host cells (Wallin et al. 2002; Schaefer et al. 2005; Scheibner et al. 2006). We showed that the cellstimulating activity of glycogen entirely depended on TLR2 and that glycogen was directly bound to TLR2 in a concentration-dependent manner. Our results indicated that glycogen is recognized by TLR2 and the recognition activates immunocytes such as macrophages to enhance the production of NO, inflammatory cytokines such as IL-6 and chemokines such as MIP-2. Furthermore, in the in vivo examination, it was shown that ESG injected into the peritoneal cavity stimulated PECs TLR2-dependently and induced the production of MIP-2 and the migration of granulocytes such as neutrophil. Taken together, these findings elucidated that TLR2 plays an important role in the recognition of glycogen. As shown in Figure 1, we indicated that glycogen induced IL-6 production from CD11b+ cells both with and without IFN-γ. On the other hand, it was shown that IFN-γ was essential for NO production from CD11b+ cells by glycogen stimulation. These findings concurred with the action profile of a TLR2 ligand, Pam3CSK4 (Figure 1). In previous studies, it TLR2 plays an important role in the recognition of glycogen Fig. 4. The cell-stimulating activity of ESG on TLR2-deficient mice PEC. PECs derived from TLR2-deficient and WT (BALB/c) mice (2 × 105 per 200 μL) were cultured with either saline, ESG (200 μg/mL), Pam3CSK4 (10 ng/mL), imiquimod (TLR7 ligand; 5 μg/mL) or LPS (100 ng/mL) in the presence of IFN-γ (10 ng/ mL). After 48 h, the culture fluid was withdrawn and the amounts of NO, IL-6 and MIP-2 in the supernatants were determined. White and black bars indicate the results for WT and TLR2KO PECs, respectively. Pam3 and IMQ in the graph indicate Pam3CSK4 and imiquimod, respectively. Values are the mean ± SD. Data are representative of three separate experiments (each experiment was performed in triplicate). ***Significantly different (P < 0.001) between WT and TLR2KO by an unpaired t-test. has been reported that the TLR4 ligand was able to induce NO production from macrophages without the addition of exogenous IFN-γ or IFN-β (Schilling et al. 2002; Toshchakov et al. 2002). These papers have clarified that TLR4 signaling enhances IFN-β production via the TRIF pathway (the MyD88-independent pathway), and the phosphorylation of STAT1 by IFN-β results in NO production from a macrophage. On the other hand, these authors have described that TLR2 ligands by itself hardly induces NO production because IFN-γ and IFN-β are poorly or not induced by TLR2 signaling that completely depends on the MyD88-dependent pathway (Schilling et al. 2002; Toshchakov et al. 2002), although another report showed that the IFNs were not needed for NO production via TLR2 activation either (Wang et al. 2006). Therefore, the addition of IFN-γ or IFN-β into the culture medium is extremely important in NO production from a macrophage by TLR2 ligands. Additionally, because IL-6 is induced via the MyD88-dependent pathway by TLR2 activation, IFN-γ and IFN-β are unnecessary for IL-6 production by TLR2 ligands. Our results in this study were substantially similar to these previous findings concerned with TLR2. These findings strongly support our contention that glycogen is recognized by TLR2. In examining the cell-stimulating mechanism of certain compounds, contamination of the specimen with LPS and lipoproteins presents a potentially important problem. Though several cell components have been suggested as novel TLR ligands, some of these proposals were later withdrawn when the apparent activity of these ligands was found to be caused by lipopeptide contamination (Bausinger et al. 2002; Lee et al. 2002; Reed et al. 2003). Thus, it is essential that efforts should be made to conclusively determine whether the putative ligands of TLRs are really the molecule under investigation or simply contaminant(s). Because glycogens extracted from natural sources have often been contaminated with LPSs, we enzymatically synthesized ultra-pure glycogen (ESG) with almost the same characteristics as natural-source glycogen (Kajiura et al. 2008, 2010). We have confirmed that only a negligible level (0.0059 ppm) of endotoxin was detected in the ESG sample by the Limulus test and that treatment with polymyxin B had no effect on the stimulating activity of ESG (Kakutani et al. 2007). Furthermore, the cellstimulating activity of ESG was completely eliminated by treatment with a glycogen-degrading enzyme such as glucoamylase (Figure 8). These results demonstrated that the macrophage-stimulating activity of ESG depends on ESG itself but not on any contaminants. Some reports have suggested that glycogen-like α-glucans from various organisms have immunostimulating activities and that TLRs and CLRs are related to the activities (Nair 151 R Kakutani et al. Fig. 5. Western blot analyses of several phosphorylated proteins in NF-κB and MAPK signaling pathways by ESG stimulation. (A) PECs (1 × 106 per 2 mL) derived from BALB/c mice were stimulated by saline, ESG (200 μg/mL) or Pam3CSK4 (10 ng/mL) in the presence of IFN-γ (10 ng/mL) for the indicated times. (B) PECs (1 × 106 per 2 mL) derived from TLR2KO and WT (BALB/c) mice were stimulated by either saline, ESG (200 μg/mL), Pam3CSK4 (10 ng/mL), imiquimod (5 μg/mL) or LPS (100 ng/mL) in the presence of IFN-γ (10 ng/mL) for 90 min. Pam3 and IMQ in the graph indicate Pam3CSK4 and imiquimod, respectively. Data are from one representative of three separate experiments. et al. 2004, 2006; Bittencourt et al. 2006; Geurtsen et al. 2009). These α-glucans were reported to be constructed from α-1,4-glucosyl chains connected with α-1,6-branch linkages resembling glycogen. However, we note significant differences between the α-glucans and glycogen in structural and functional properties. For example, α-glucan prepared from the medicinal plant Tinospora cordifolia was found to activate macrophages (Nair et al. 2006). The α-glucan also activated lymphocytes such as natural killer (NK) cells, T cells and B cells (Nair et al. 2004). Unlike glycogen, its activity was 152 reported to involve TLR6 but not TLR2 (Nair et al. 2006). Another α-glucan derived from the cell surface of a fungus, Pseudallescheria boydii, induced cytokine secretion by cells of the innate immune system with a mechanism that involved TLR2 (Bittencourt et al. 2006). Structurally, this α-glucan has a much higher degree of branching (24%) than glycogen (around 11%). In addition, other extracellular α-1,4/1,6 glucan derived from a pathogenic mycobacterium, Mycobacterium tuberculosis, activated immunocytes through DC-SIGN (Geurtsen et al. 2009). Another report suggested that this TLR2 plays an important role in the recognition of glycogen Fig. 6. The cell-stimulating activity of ESG toward TLR2/4DKO and MyD88KO PECs. PECs (2 × 105 per 200 μL) derived from TLR2/4DKO, MyD88KO and WT mice were incubated with either saline, ESG (200 μg/mL), Pam3CSK4 (10 ng/mL), imiquimod (5 μg/mL) or LPS (100 ng/mL) in the presence of IFN-γ (10 ng/mL). After 48 h, the culture fluid was withdrawn and NO, IL-6 and MIP-2 in the supernatant was measured. The results of TLR2/4DKO and WT (C57BL/6) PECs are shown in (A)–(C), and the results of MyD88KO and WT (BALB/c) PECs are shown in (D)–(F). Pam3 and IMQ in the graph indicate Pam3CSK4 and imiquimod, respectively. Values are the mean ± SD. Data are representative of two independent experiments (each experiment was performed in triplicate). ***Significantly different (P < 0.001) between WT and KO mice by an unpaired t-test. α-glucan has a more compact structure than glycogen because of differences in backbone chain lengths and threedimensional structures (Dinadayala et al. 2008). As regard the intracellular glycogen in microorganisms, it was recently reported that the glycogen accumulation in a pathogenic bacterium, Chlamydia trachomatis, seemed to be related to the TLR2 activation of host cells (O’Connell et al. 2011). The paper showed that C. trachomatis mutant lacking the ability of glycogen accumulation displayed the reduction in TLR2 activation compared with WT. Similarly, the glucose limitation of the bacterium resulted in a lower level of glycogen and a significantly reduced level of TLR2 activation. However, there is no clear evidence to show that the intracellular glycogen directly related to TLR2 activation. To our best knowledge, ESG is the only α-glucan of which direct binding to its receptor has been demonstrated. Furthermore, the structural properties of ESG such as molecular weight and chain length can be controlled. Thus, ESG could be a useful tool to investigate the relationship between the structures of α-glucans and their immunomodulating activities. Besides TLRs, it is known that CLRs are related to the potentiating of innate immunity (Saijo et al. 2007; Yamasaki et al. 2008; den Dunnen et al. 2009). For example, dectin-1 is a glycoprotein CLR for β-1,3 glucans, and this receptor is 153 R Kakutani et al. Fig. 7. Relationship between ESG stimulation and various TLRs. HEK293 reporter cells expressing each TLR and control cells were cultured with either saline, ESG (500 μg/mL) or the respective positive control ligands for 16 h, and NF-κB activities were determined. Details of positive control ligands are given in Materials and methods. Data are represented as the mean ± SD from two independent experiments (each experiment was performed in duplicate). *Significantly different (P < 0.05) from negative control saline by an unpaired t-test. Fig. 8. Abolition of the immunostimulating activity of ESG by glucoamylase treatment. Either saline, ESG (200 μg/mL), Pam3CSK4 (10 ng/mL) or FSL-1 (100 ng/mL) treated with/without glucoamylase (GA) were cultured with RAW264.7 (1 × 105 per 200 μL). After 48 h, NO production in the broth was determined by the Griess reagent. White and black bars indicate the results with and without GA, respectively. Values are the mean ± SD. Data are representative of three separate experiments (each experiment was performed in triplicate). ***Significantly different (P < 0.001) vs GA-treated specimens by an unpaired t-test. assumed to be involved in recognizing pathogenic fungi (Adachi et al. 2004; Saijo et al. 2007). In addition, dectin-1 accelerates the TLR2-induced NF-κB activation by Zymosan, while this receptor independently activates the Syk-mediated signaling pathway unaided by TLR2-mediated signaling (Ikeda et al. 2008). We examined whether glycogen is recognized by dectin-1 besides TLR2. As a result, dectin-1 was bound not to glycogen but to β-glucans such as SPG and OX-CA, whereas TLR2 was bound not to these β-glucans but to glycogen (Figure 9C). These findings revealed that dectin-1 is not related to the recognition of glycogen. Although both glycogen and β-glucans are glucose polymers with high molecular weight, the mechanism of glycogen recognition by a macrophage was entirely different from that of β-glucan recognition. Dectin-1 strictly recognizes the structural difference between α- and β-glucosyl bonds in several glucans. 154 Fig. 9. The binding activities of TLR2 to glycogen. (A) Bindings of ESG (100–1000 μg/mL) to TLR2 (2.5 μg/mL) were tested by ELISA. Pam3CSK4 (100 μg/mL), LPS (500 μg/mL) or imiquimod (500 μg/mL) was used as a control compound. Pam3 and IMQ in the graph indicate Pam3CSK4 and imiquimod, respectively. (B) Dose response binding curve at the indicated concentration of TLR2 to ESG (500 μg/mL). (C) Bindings of ESG to dectin-1 (β-glucan receptor) and comparison with other glucans such as oyster glycogen (natural-source glycogen; α-1,4/1,6 glucan), soluble starch (α-1,4/ 1,6 glucan), dextran (α-1,6 glucan), SPG (soluble β-1,3/1,6 glucan) and OX-CA ( particle β-1,3/1,6 glucan). All glucans, Pam3CSK4, TLR2 and dectin-1 were tested with concentrations of 1000, 100, 2.5 and 1 μg/mL, respectively. OysGly and Pam3 in the graph indicate oyster glycogen and Pam3CSK4, respectively. Data are represented as the mean ± SD from three independent experiments (each experiment was performed in quadruplicate). **Significantly different (P < 0.01) from the negative control by one-way ANOVA followed by Dunnett’s multiple comparison test. In this paper, we demonstrated that glycogen activates immunocyte such as macrophages via TLR2 existing on the cell surface to produce the inflammatory cytokines. It was TLR2 plays an important role in the recognition of glycogen Fig. 10. Effect of ESG intraperitoneal injection on PECs in WT (BALB/c) and TLR2KO mice. (A–C) Either saline, ESG (500 μg) or Pam3CSK4 (50 ng) was injected into the peritoneal cavities of WT and TLR2KO mice. Several PECs were retrieved on the day after injection, and white blood cell number (A) and CD11b+Gr-1+ cell populations (B and C) were determined. (D) WT and TLR2KO mice were injected intraperitoneally with either saline, ESG (250 μg) or Pam3CSK4 (250 ng). After 5 h, MIP-2 concentration in each peritoneal lavage fluid was determined by ELISA. Black and white bar in the graph indicate WT and TLR2KO mice, respectively. Values are the mean ± SE. Several significant differences were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test or an unpaired t-test (**P < 0.01). indicated that glycogen functions as an immunostimulator to activate the immune cell. It is well known that glycogen exists in various organisms such as microorganism and mammals (Geddes 1986; Brown 2004; Dinadayala et al. 2008; O’Connell et al. 2011). It should be a rewarding area of research to elucidate the immunological function of glycogen. 155 R Kakutani et al. Materials and methods Chemicals LPS (Escherichia coli 055:B5), RPMI-1640, Dulbecco’s modified Eagle’s medium and Hank’s balanced salt solution (HBSS) were obtained from Sigma Chemical Co. (St Louis, MO). Murine recombinant IFN-γ, SPG and phosphatebuffered saline (PBS) were purchased from Becton Dickinson (San Jose, CA), Kaken Pharmaceutical Co. (Tokyo, Japan) and GIBCO (Carlsbad, CA), respectively. Pam3CSK4, LPS-EB, FSL-1, Poly I:C, imiquimod and CpG-ODN were obtained from InvivoGen Co. (San Diego, CA). ESG with an average molecular weight of 5.0 × 106 was prepared as described previously (Kajiura et al. 2008). When it is assumed that ESG is a defined molecule, the amounts of ESG (Mr; 5.0 × 106, 200 μg/mL) and Pam3CSK4 (Mr; 1.5 × 103, 10 ng/mL) used in the cell-stimulating assays are calculated 40 and 6.7 nM, respectively. Cell lines and animals RAW264.7 and HEK293 cells were obtained from the RIKEN Cell Bank (Tsukuba, Japan). TLR2KO, TLR4KO, MyD88KO mice (BALB/c background) and TLR2/4DKO mice (C57BL/6 background) between 6 and 9 weeks of age were purchased from Oriental Bio Service Co. (Kyoto, Japan). BALB/c and C57BL/6 mice between 6 and 9 weeks of age were purchased from Japan SLC (Shizuoka, Japan). All animal experiments were approved by the Institutional Animal Care and Use Committee of Ezaki Glico Co. Ltd. and performed in accordance with the Guidelines for Proper Conduct of Animal Experiments (Science Council of Japan). Preparation of CD11b+ cells CD11b+ cells were prepared from PECs of BALB/c mice by MACS. Briefly, PECs were retrieved from resident BALB/c mice with cold HBSS (5 mL). These cells (2 × 107 cells) were added with mouse CD11b MicroBeads (Miltenyi Biotec Co., Bergisch Gladbach, Germany) and incubated at 4°C for 15 min. After washing, CD11b+ and CD11b− cell groups were separated with using MACS Column (Miltenyi Biotec Co.) and MACS Separator (Miltenyi Biotec Co.). CD11b+ rates of obtained CD11b+ and CD11b− cell groups were >90 and <1%, respectively. Cell stimulation and determination of NO, TNF-α, IL-6 and MIP-2 Several mice PECs, CD11b+ and CD11b− cells separated from PECs and RAW264.7 cells were plated in 24- or 96-well plates and cultured with several stimuli in the absence or the presence of IFN-γ for the indicated times. The culture fluids were retrieved and the amounts of NO, TNF-α, IL-6 and MIP-2 in the supernatants were determined as described previously (Ohno et al. 1996; Harada et al. 2002). NO production was measured by using the Griess reagent. TNF-α, IL-6 and MIP-2 concentrations were determined by BD OptEIA Mouse TNF ELISA Set (Becton Dickinson), BD OptEIA Mouse IL-6 ELISA Set (Becton Dickinson) and DuoSet CXCL2/MIP-2 kit (R&D systems, Minneapolis, MN), respectively. 156 Reporter gene assay HEK293 cells (3 × 104/100 μL) were cultured without antibiotics in a 96-well plate for 16 h. These cells were transfected with reporter gene plasmids and TLR expression plasmids using Lipofectamine LTX and Plus reagents (Invitrogen, Carlsbad, CA, USA). Murine tlr2, tlr4, md-2 and cd14 genes were cloned into pDisplay (Invitrogen), p3xFLAG-CMV-14 (Invitrogen), pBudCE4.1 (Invitrogen) and pcDNA3.1 (Invitrogen), respectively. In the experiment on TLR4-mediated NF-κB activation, each well received 50 ng of pNF-κB-Luc reporter plasmid (Stratagene, La Jolla, CA, U.S.A.) and 10 ng of pRL-TK control reporter plasmid (Promega, WI), together with 25 ng mTLR4, 10 ng mMD-2 and 10 ng mCD14 expression vectors. In the experiment on TLR2-mediated NF-κB activation, each well received 50 ng of pNF-κB-Luc reporter plasmid and 10 ng of pRL-TK control reporter plasmid, together with 25 ng of TLR2 expression vector. These cells were incubated for 24 h after lipofection. ESG, LPS, Pam3CSK4 or CpG-ODN was added to TLR4/MD-2/CD14- or TLR2-expressing reporter cells and incubated for 24 h. After the cells were lysed, firefly and Renilla luciferase activities were determined by the Dual-Luciferase Reporter Assay System (Promega) and a luminometer, Centro LB 960 (Berthold, Germany). Cell-stimulating activity by several specimens was expressed as the ratio of pNF-κB-Luc (firefly) activity to RL-TK (Renilla) activity. Ultra-pure LPS (LPS-EB) instead of conventional grade LPS was used for these reporter assays. Real-time PCR Stimuli-treated cells were washed with PBS, and total RNAs were isolated from these cells using QIAshredder and RNeasy Mini Kit (QIAGEN, Germantown, MD). Total RNAs were converted to cDNA by a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). cDNA was mixed with Power SYBR Green PCR master mix (Applied Biosystems) and primers for TNF-α gene designed by utilizing Primer Express Software, and levels of gene expression were determined by a 7500 Fast Real-Time PCR System (Applied Biosystems). Western blot analysis PECs from several mice were plated onto a 6-well plate at 1 × 106 cells in 2 mL/well and treated with either saline, ESG (200 μg/mL), Pam3CSK4 (10 ng/mL), imiquimod (5 μg/mL) or LPS (100 ng/mL) in the presence of IFN-γ (10 ng/mL) for the indicated times. These cells were washed with PBS, then lysed in 3×SDS sample Buffer Blue (Cell Signaling Technology, Beverly, MA) and sonicated. The lysates were fractionated by 10% SDS–PAGE. After electrophoresis, proteins were transferred to PVDF membranes, and the membranes were incubated in blocking buffer [1% casein sodium in tris buffered saline (TBS) plus 0.05% Tween-20 (TBST)]. The membranes were incubated at 4°C overnight in TBST containing the primary antibody, washed for 30 min in TBST, incubated for 1 h in the secondary antibody and washed for another 30 min in TBST. The membranes were treated with ECL-plus substrate solution (GE Healthcare, Buckinghamshire, UK) and incubated under dark conditions TLR2 plays an important role in the recognition of glycogen for 5 min at room temperature. Spots on these membranes were detected using a LAS-4000UVmini (Fuji-film, Tokyo, Japan). In this experiment, primary antibodies for ERK1/2 ( p44/42 MAPK), phospho-ERK1/2, p38 MAPK, phosphop38, SAPK/JNK, phospho-SAPK/JNK, phospho-IκB-α, phospho-NF-κB p65, phospho-Akt and glyceraldehyde-3phosphate dehydrogenase (GAPDH) (Cell Signaling Technology) were used. TLR screening To investigate the interaction of ESG toward other TLRs than TLR2 and TLR4, several TLR-expressing HEK293 cells transfected with pNifty-SEAP reporter plasmid (InvivoGen Co.) were used. Either saline, ESG (500 μg/mL) or the respective positive control ligands were added to each reporter cell and incubated at 37°C for 16 h. The activity of SEAP (secreted form of human placental alkaline phosphatase) induced by NF-κB was examined after incubation. Heat-killed Listeria monocytogenes (108 cells/mL), Poly I:C (1 μg/mL), LPS (1 μg/mL), flagellin (100 ng/mL), CL097 (1 μg/mL), CL075 (10 μg/mL) plus PolydT (10 μM), CpG-ODN 1826 (1 μg/mL) and TNF-α (1 μg/mL) were used as positive control ligands toward TLR2, 3, 4, 5, 7, 8 and 9 and NF-κB (control)-expressing cells, respectively. Treatment with glucoamylase ESG (200 μg) was incubated with 5 U/mL glucoamylase (EC 3.2.1.3; TOYOBO, Osaka, Japan), the enzyme that cleaves α-1,4- and α-1,6-glucosidic bonds, in 0.02 M sodium acetate buffer ( pH 3.5) at 40°C for 16 h. It was confirmed that glycogen was quantitatively converted to glucose by this treatment. These mixtures were centrifuged, then the supernatants were added to RAW264.7 cells plated 1 × 105 cells per 0.2 mL and incubated in the presence of IFN-γ (10 ng/ mL) at 37°C for 48 h. NO concentrations in culture supernatants were determined by the above-mentioned method. In addition, to confirm that glucoamylase is not able to resolve the TLR2 ligands, Pam3CSK4 and FSL-1 were used as control ligands. ESG bindings to TLR2 and dectin-1 Binding of ESG to each receptor was tested as follows. A 96-well plate (Costar Co., Cambridge, MA, USA) was coated with a monoclonal immunoglobulin M (IgM) antibody against glycogen (Baba 1993) at 4°C for 16 h. The plate was washed with PBS containing 0.05% Tween-20 and blocked with PBS containing 1% bovine serum albumin at 37°C for 1 h. It was then washed and treated with 100–1000 μg/mL of ESG at 37° C for 2 h. After washing, the plate was incubated with 0.15-20 μg/mL of purified recombinant mouse TLR2 (R&D Systems) at 37°C for 2 h. It was then treated with 0.5 μg/mL of a biotinylated anti-mouse TLR2 antibody (R&D Systems) at 37°C for 1 h and thereafter incubated with peroxidaseconjugated streptavidin (Becton Dickinson) at 37°C for 1 h. Finally, the plates were developed with a TMB substrate system (KPL, Inc., MD). The development of color was stopped with 1 N phosphoric acid and absorbencies at 450/ 630 nm were determined. Meanwhile, bindings of ESG to dectin-1 were evaluated by using dectin-1/Fc (Tada et al. 2008) or control/Fc. Dectin-1/Fc was detected by hIgG-HRP (Bethyl Co., Montgomery, TX) and finally determined by the TMB system. To compare ESG with natural-source glycogen, glycogen prepared from oyster was used. Pam3CSK4 (100 μg/ mL), LPS (500 μg/mL), imiquimod (500 μg/mL), dextrin (soluble starch; α-1,4/1,6 glucan; 1000 μg/mL), dextran (α-1,6 glucan; 1000 μg/mL), SPG (soluble β-1,3/1,6 glucan; 1000 μg/ mL) and OX-CA ( particle β-1,3/1,6 glucan; 1000 μg/mL) (Ohno et al. 1999) were used as a positive/negative control without the coating of an anti-glycogen antibody. In vivo examination TLR2KO and WT mice were injected intraperitoneally with ESG (500 μg), Pam3CSK4 (50 ng) or saline, and PECs were prepared from these mice on the day after injection. The number of each PEC was determined by using hemocytometer. CD11b+Gr-1+ cell populations of these PECs were detected as described previously (Harada et al. 2002). Briefly, PECs were washed with PBS and added anti-mouse CD16/ CD32 FcBlock™ (Becton Dickinson) for blocking FcR-mediated binding of the monoclonal antibodies (mAb). Thereafter, PECs were stained with anti-mouse CD11b-FITC (Beckman Coulter Inc., Miami, FL) and anti-mouse Gr-1-PE (Beckman Coulter Inc.) at 4°C for 1 h. The cells were washed with PBS and analyzed on Cytomics FC 500 (Beckman Coulter Inc.). Additionally, the amount of MIP-2 in the peritoneal cavity after ESG injection was determined. TLR2KO and WT mice were injected intraperitoneally with ESG (250 μg), Pam3CSK4 (250 ng) or saline, and PECs for 5 h after injection were retrieved with cold HBSS (1 mL). After centrifugation, MIP-2 concentration in each peritoneal lavage supernatant was determined by ELISA. Statistical analysis Statistical analysis was performed between two groups using unpaired t-test. Comparisons with more than three groups were analyzed by one-way analysis of variance (ANOVA) with appropriate post hoc test. A difference between groups of P < 0.05 was considered significant. Funding This work was supported in part by grants from the “Program to develop new technology to promote the agriculture, forestry, fisheries and food industries through collaboration among industry, academia and the government” and “Research and development projects to promote new policies in agriculture, forestry and fisheries” of the Ministry of Agriculture, Forestry and Fisheries, Japan. Acknowledgements We thank Drs. Otto Baba and Tatsuo Terashima (Tokyo Medical and Dental University, Tokyo, Japan) for providing us the monoclonal anti-glycogen antibody. 157 R Kakutani et al. Conflict of interest None declared. Abbreviations ANOVA, analysis of variance; CD, cluster of differentiation; CLR, C-type lectin receptor; DKO, double KO; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated kinase; ESG, enzymatically synthesized glycogen; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HBSS, Hank’s balanced salt solution; HEK293, human embryonic kidney 293; IFN-γ, interferon-γ; IgM, immunoglobulin M; IL, interleukin; KO, knock out; LPS, lipopolysaccharide; mAb, monoclonal antibodies; MACS, magneticactivated cell sorting; MAPK, mitogen-activated protein kinase; MIP-2, macrophage inflammatory protein-2; MyD88, myeloid differentiation factor 88; NF-κB, nuclear factor-kappa B; NK, natural killer; NO, nitric oxide; NOS2, nitric oxide synthase 2; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PEC, peritoneal exude cell; pNF-κB-Luc, pNF-κB luciferase; SPG, schizophyllan glucan; TBS, tris buffered saline; TBST, TBS with Tween-20; TLR, Toll-like receptor; TNF-α, tumor necrosis factor-α; WT, wild type. References Adachi Y, Ishii T, Ikeda Y, Hoshino A, Tamura H, Aketagawa J, Tanaka S, Ohno N. 2004. Characterization of β-glucan recognition Site on C-type lectin, dectin 1. Infect Immun. 72:4159–4171. Aliprantis AO, Yang RB, Mark MR, Suggett S, Devaux B, Radolf JD, Klimpel GR, Godowski P, Zychlinsky A. 1999. Cell activation and apoptosis by bacterial lipoproteins through Toll-like receptor-2. Science. 285:736–739. Baba O. 1993. Production of monoclonal antibody that recognizes glycogen and its application for immunohistochemistry. Kokubyo Gakkai Zasshi. 60:264–287. Bausinger H, Lipsker D, Ziylan U, Manie S, Briand JP, Cazenave JP, Muller S, Haeuw JF, Davanat C, de la Salle H, et al. 2002. Endotoxin-free heat shock protein 70 fails to induce APC activation. Eur J Immunol. 32:3708–3713. Beg AA. 2002. Endogenous ligands of Toll-like receptors: Implications for regulating inflammatory and immune responses. Trends Immunol. 23:509–512. Bittencourt VC, Figueiredo RT, Silva RB, Mourao-Sa DS, Fernandez PL, Sassaki GL, Mulloy B, Bozza MT, Barreto-Bergtor E. 2006. An α-glucan of Pseudallescheria boydii is involved in fungal phagocytosis and Toll-like receptor activation. J Biol Chem. 281:22614–22623. Blows JM, Calder PC, Geddes R, Wills PR. 1988. The structure of placental glycogen. Placenta. 9:493–500. Brown AM. 2004. Brain glycogen re-awakened. J Neurochem. 89:537–552. Campos MA, Almeida IC, Takeuchi O, Akira S, Valente EP, Procopio DO, Travassos LR, Smith JA, Golenbock DT, Gazzinelli RT. 2001. Activation of Toll-like receptor-2 by glycosylphosphatidylinositol anchors from a protozoan parasite. J Immunol. 167:416–423. Chamaillard M, Hashimoto M, Horie Y, Masumoto J, Qiu S, Saab L, Ogura Y, Kawasaki A, Fukase K, Kusumoto S, et al. 2003. An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat Immunol. 4:702–707. den Dunnen J, Gringhuis SI, Geijtenbeek TB. 2009. Innate signaling by the C-type lectin DC-SIGN dictates immune responses. Cancer Immunol Immunother. 58:1149–1157. Dinadayala P, Sambou T, Daffe M, Lemassu A. 2008. Comparative structural analyses of the α-glucan and glycogen from Mycobacterium bovis. Glycobiology. 18:502–508. 158 Geddes R. 1986. Glycogen: A metabolic viewpoint. Biosci Rep. 6: 415–428. Geurtsen J, Chedammi S, Mesters J, Cot M, Driessen NN, Sambou T, Kakutani R, Ummels R, Maaskant J, Takata H, et al. 2009. Identification of mycobacterial α-glucan as a novel ligand for DC-SIGN: Involvement of mycobacterial capsular polysaccharides in host immune modulation. J Immunol. 183:5221–5231. Greenberg CC, Jurezak MJ, Danos AM, Brady MJ. 2006. Glycogen branches out: New perspectives on the role of glycogen metabolism in the integration of metabolic pathways. Am J Physiol Endocrinol Metab. 291: E1–E8. Harada T, Miura N, Adachi Y, Nakajima M, Yadomae T, Ohno N. 2002. Effect of SCG, 1,3-β-D-glucan from Sparassis crispa on the hematopoietic response in cyclophosphamide induced leukopenic mice. Biol Pharm Bull. 25:931–939. Harmon CS, Phizackerley PJ. 1983. The effects of starvation and re-feeding on glycogen metabolism in mouse tail skin. Biochem J. 212:679–683. Ikeda Y, Adachi Y, Ishii T, Miura N, Tamura H, Ohno N. 2008. Dissociation of Toll-like receptor 2-mediated innate immune response to zymosan by organic solvent-treatment without loss of dectin-1 reactivity. Biol Pharm Bull. 31:13–18. Iwasaki A, Medzhitov R. 2004. Toll-like receptor control of the adaptive immune responses. Nat Immunol. 5:987–995. Kajiura H, Kakutani R, Akiyama T, Takata H, Kuriki T. 2008. A novel enzymatic process for glycogen production. Biocatal Biotransform. 26:133–140. Kajiura H, Takata H, Kuriki T, Kitamura S. 2010. Structure and solution properties of enzymatically synthesized glycogen. Carbohydr Res. 345:817–824. Kakutani R, Adachi Y, Kajiura H, Takata H, Kuriki T, Ohno N. 2007. Relationship between structure and immunostimulating activity of enzymatically synthesized glycogen. Carbohydr Res. 342:2371–2379. Kakutani R, Adachi Y, Kajiura H, Takata H, Kuriki T, Ohno N. 2008. Stimulation of macrophage by enzymatically synthesized glycogen: The relationship between structure and biological activity. Biocatal Biotransform. 26:152–160. Lee HK, Lee J, Tobias PS. 2002. Two lipoproteins extracted from Escherichia coli K-12 LCD25 lipopolysaccharide are the major components responsible for Toll-like receptors. J Immunol. 168:4012–4017. Nair PK, Melnick SJ, Ramachandran R, Escalon E, Ramachandran C. 2006. Mechanism of macrophage activation by (1,4)-α-D-glucan isolated from Tinospora cordifolia. Int Immunopharmacol. 6:1815–1824. Nair PK, Rodriguez S, Ramachandran R, Alamo A, Melnick SJ, Escalon E, Gareia PI, Jr, Wnuk SF, Ramachandran C. 2004. Immune stimulating properties of a novel polysaccharide from the medicinal plant Tinospora cordifolia. Int Immunopharmacol. 4:1645–1659. O’Connell CM, AbdelRahman YM, Green E, Darville HK, Saira K, Smith B, Darville T, Scurlock AM, Meyer CR, Belland RJ. 2011. Toll-like receptor 2 activation by Chlamydia trachomatis is plasmid dependent, and plasmid-responsive chromosomal loci are coordinately regulated in response to glucose limitation by C. trachomatis but not by C. muridarum. Infect Immun. 79:1044–1056. Ohno N, Egawa Y, Hashimoto T, Adachi Y, Yadomae T. 1996. Effect of β-glucans on the nitric oxide synthesis by peritoneal macrophage in mice. Biol Pharm Bull. 19:608–612. Ohno N, Uchiyama M, Tsuzuki A, Tokunaka K, Miura NN, Adachi Y, Aizawa MW, Tamura H, Tanaka S, Yadomae T. 1999. Solubilization of yeast cell-wall β-(1 → 3)-D-glucan by sodium hypochlorite oxidation and dimethyl sulfoxide extraction. Carbohydr Res. 316:161–172. Reed RC, Berwin B, Baker JP, Nicchitta CV. 2003. GRP94/gp96 elicits ERK activation in murine macrophages: A role of endotoxin contamination in NFκB activation and nitric oxide production. J Biol Chem. 278:31853–31860. Saijo S, Fujikado N, Furuta T, Chung SH, Kotaki H, Seki K, Sudo K, Akira S, Adachi Y, Ohno N, et al. 2007. Dectin-1 is required for host defense against Pneumocystis carinii but not against Candida albicans. Nat Immunol. 8:39–46. Salmoral EM, Tolmasky DS, Krisman CR. 1990. Evidence for the presence of glycogen in rat thymus. Cell Mol Biol. 36:163–174. Schaefer L, Babelova A, Kiss E, Hausser HJ, Baliova M, Krzyzankova M, Marsche G, Young MF, Mihalik D, Gotte M, et al. 2005. The matrix component biglycan is proinflammatory and signals through Toll-like receptors 4 and 2 in macrophages. J Clin Invest. 115:2223–2233. TLR2 plays an important role in the recognition of glycogen Scheibner KA, Lutz MA, Boodoo S, Fenton MJ, Powell JD, Horton MR. 2006. Hyaluronan fragments acts as an endogenous danger signal by engaging TLR2. J Immunol. 177:1272–1281. Schilling D, Thomas K, Nixdorff K, Vogel SN, Fenton MJ. 2002. Tolllike receptor 4 and Toll-IL-1 receptor domain-containing adapter protein (TIRAP)/myeloid differentiation protein 88 adapter-like (Mal) contribute to maximal IL-6 expression in macrophage. J Immunol. 169: 5874–5880. Schwandner R, Dziarski R, Wesche H, Rothe M, Kirschning CJ. 1999. Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by Toll-like receptor 2. J Biol Chem. 274:17406–17409. Scott RB, Still WJ. 1968. Glycogen in human peripheral blood leukocytes. II. The macromolecular state of leukocyte glycogen. J Clin Invest. 47:353–359. Tada R, Adachi Y, Ishibashi K, Tsubaki K, Ohno N. 2008. Binding capacity of a barley β-D-glucan to the β-glucan recognition molecule dectin-1. J Agric Food Chem. 56:1442–1450. Taegtmeyer H. 2004. Glycogen in the heart – an expanded view. J Mol Cell Card. 37:7–10. Takaya Y, Uchisawa H, Ichinohe H, Sasaki J, Ishida K, Matsue H. 1998. Antitumor glycogen from scallops and the interrelationship of structure and antitumor activity. J Mar Biotechnol. 6:208–213. Takeda K, Kaisho T, Akira S. 2003. Toll-like receptors. Annu Rev Immunol. 21:335–376. Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, Takeda K, Akira S. 1999. Differential roles of TLR2 and TLR4 in recognition of Gram-negative and Gram-positive bacterial cell wall components. Immunity. 11:443–451. Thoma-Uszynski S, Stenger S, Takeuchi O, Ochoa MT, Engele M, Sieling PA, Barnes PF, Rollinghoff M, Bolcskei PL, Wagner M, et al. 2001. Induction of direct antimicrobial activity through mammalian Toll-like receptors. Science. 291:1544–1547. Toshchakov V, Jones BW, Perera PY, Thomas K, Cody MJ, Zhang S, Williams BRG, Major J, Hamilton TA, Fenton MJ, et al. 2002. TLR4, but not TLR2, mediates IFN-β-induced STAT1α/β-dependent gene expression in macrophages. Nat Immunol. 3:392–398. Wallin RP, Lundqvist A, More SH, von Bonin A, Kiessling R, Ljunggren HG. 2002. Heat-shock proteins as activators of the innate immune system. Trends Immunol. 23:130–135. Wang Q, McLoughlin RM, Cobb BA, Charrel-Dennis M, Zaleski KJ, Golenbock D, Tzianabos AO, Kasper DL. 2006. A bacterial carbohydrate links innate and adaptive responses through Toll-like receptor 2. J Exp Med. 203:2853–2863. Yamasaki S, Ishikawa E, Sakuma M, Hara H, Ogata K, Saito T. 2008. Mincle is an ITAM-coupled activating receptor that senses damaged cells. Nat Immunol. 9:1179–1188. Yoneyama M, Fujita T. 2007. Function of RIG-I-like receptors in antiviral innate immunity. J Biol Chem. 282:15315–15318. 159