Download Essential role of Toll-like receptor 2 in

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
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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
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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
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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+
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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
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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
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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
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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
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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.
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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.
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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.
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