From the Unité de Pathogénie Microbienne
Moléculaire, INSERM U389, ¶ Unité de Pathogénie
Bactérienne des Muqueuses, ** Groupe d'Immunité
Innée et Signalisation, Institut Pasteur, 28, Rue du Dr. Roux,
75724 Paris Cedex 15, France and the
INSERM
U434, Fondation Jean
Dausset/CEPH, Paris, France
Received for publication, November 21, 2002, and in revised form, January 13, 2003
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ABSTRACT |
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Nod2 activates the NF- Crohn's disease is a chronic inflammatory disorder of the
gastrointestinal tract. The development of this disease is known to be
influenced by both environmental factors and genetic predisposition. A
significant advance in the understanding of this disease was achieved
by the identification of Nod2/CARD15 as the first
susceptibility gene for Crohn's disease in Caucasian populations (1,
2). Nod2 belongs to the recently described family of intracellular Nod
proteins, which contain a nucleotide-binding site domain flanked by a leucine-rich repeat domain (3, 4). Among this family, proteins
such as Nod1/CARD4 and Nod2 detect bacterial products to induce the
activation of proinflammatory signaling pathways, such as the NF- Since bacterial products have been shown to activate Nod2 (2, 6), the
implication of Nod2 in Crohn's disease may support the hypothesis that
enteric bacteria-host interactions could have an etiological role in
this disorder. However, the precise characterization of the bacterial
motifs detected by Nod2 remains to be addressed. In this study, we
aimed to examine the sensitivity of Nod2 toward different
peptidoglycans (PGNs),1 a
cell wall product common to both Gram-positive and Gram-negative bacteria. Our results show that Nod2 senses muramyl dipeptide (MDP),
which is the minimal PGN motif common to both Gram-positive and
Gram-negative bacteria. These findings impact on our current understanding of the etiology of Crohn's disease. Additionally, these
data will contribute to the elucidation of the mode of action of MDP,
an immunomodulatory agent used for decades in adjuvant preparations.
Preparation of Highly Purified Peptidoglycans from Gram-negative
and Gram-positive Bacteria--
Bacterial strains used to prepare PGN
were as follows: Escherichia coli K12, Shigella
flexneri 5a M90T (wild type), and Bacillus subtilis
168, Staphylococcus aureus COL (from Olivier Chesneau, Institut Pasteur). PGNs of E. coli and S. flexneri were purified as described by Glauner et al.
(11). PGNs of B. subtilis and S. aureus were
purified as described by de Jonge et al. (12). Briefly,
bacteria were harvested in exponential growth phase at an optical
density (600 nm) of 0.4-0.6 and quickly chilled in an ice-ethanol bath
to minimize PGN hydrolysis by endogeneous autolysins. Pellets were
resuspended in ice-cold water and added drop by drop to 8% SDS
boiling. Samples were boiled for 30 min, allowing immediate
inactivation of autolysins. Polymeric PGN, which is insoluble, was
recovered by centrifugation and washed several times until no SDS could
be detected. SDS assay was done as described by Hayashi (13). SDS
treatment removes contaminating proteins, non-covalently bound
lipoproteins and LPS. Gram-positive samples were physically broken with
acid washed glass beads (<100 nm). The PGN fraction was recovered by
differential centrifugation to remove cellular debris. All PGNs were
further treated with Cells and Reagents--
HEK293T and HeLa epithelial cells, and
RAW macrophage cells, were cultured in Dulbecco's modified Eagle's
medium containing 10% fetal calf serum. Prior to transfection, HEK293T
cells were seeded into 24-well plates at a density of 1 × 105 cells/ml as described previously (5). MDP LD and MDP LL
were from Calbiochem and reported to be 98% pure by TLC. LPS was from E. coli O111:B4 (Sigma), commercial S. aureus PGN
was from Fluka, and TNF Expression Plasmids and Transient Transfections--
The
expression plasmid for FLAG-tagged Nod1 was from Gabriel Nuñez
(University of Michigan Medical School, Ann Arbor, MI) and has been
described previously (15). For Nod2, a peripheral blood leukocyte
cDNA library ( NF-
For immunofluorescence studies, NF- Nod2 Senses Peptidoglycans from Gram-negative and Gram-positive
Bacteria--
Previous studies demonstrated that Nod2 can be activated
by commercial PGN from Staphyloccocus aureus and LPSs from
various Gram-negative bacteria (2, 6). We aimed to investigate the sensing specificity of Nod2 toward PGNs from Gram-negative or Gram-positive bacteria. Indeed, even though Gram-negative and Gram-positive bacteria contain PGN, its organization and structure differ substantially between these two groups (17). Therefore, PGNs
from E. coli, S. flexneri, B. subtilis, and S. aureus were purified according to
experimental procedures specifically designed for Gram-positive or
Gram-negative bacteria (11, 12). These harsh purification steps are
well suited for the elimination of the possible contaminants, such as
LPS (through SDS treatments), lipoproteins (through SDS and trypsin
treatments), lipoteichoic acid (LTA), teichoic acid (TA), capsule
(through HF treatment of Gram-positive PGN). Indeed, amino acid and
saccharide analysis of the purified PGNs following HPLC revealed the
absence of amino acids or amino sugars other than those related to PGN,
thus excluding the possibility that contaminant polypeptidic residues
remained at the end of our purification procedures (data not shown).
Equivalent amounts of purified PGNs (1 µg) were then added to cells
transiently transfected with Nod2, and the subsequent activation of the
NF- Nod2 Mediates PGN Sensing through MDP Detection--
These
observations prompted us to define what is the minimal PGN compound
that would be common to both Gram-negative and Gram-positive bacteria
and could activate Nod2. The sugar backbone of PGN is made of chains of
repeating disaccharide units composed of N-acetylglucosamine (GlcNAc)
MDP has been studied extensively in the past 2 decades because of its
immunostimulatory properties (19). It is also well documented that the
biological activity of this molecule is completely abolished if its
second amino acid, D-Glx, is replaced by the L-Glx enantiomer (MDP LL). We therefore investigated
whether Nod2 could detect the biologically inactive MDP LL to the same
extent as MDP. Chemically synthesized MDP LL (1 µg) was then added to Nod2-transfected cells, and in these conditions we detected no synergistic activation of the Nod2-dependent NF- Macrophages but Not Epithelial Cells Can Detect MDP--
In
contrast to Nod1, which is expressed in virtually all adult tissues
(15, 20), the expression of Nod2 is predominantly in monocytes derived
from peripheral blood leukocytes (21). In epithelial cells, Nod2
expression remains below detection limits in basal conditions (Ref. 22
and data not shown). Therefore, we took advantage of this restricted
expression profile to compare the sensitivity of epithelial cells and
macrophages toward MDP stimulation. To this end, macrophages or
epithelial cells were stimulated with MDP LD or MDP LL, and the
activation of the NF- Defective PGN or MDP Sensing in Crohn's Disease-associated Nod2
Variant--
Following the discovery of Nod2 as a
susceptibility gene for Crohn's disease, a critical issue is now to
understand how mutations that affect Nod2 can account for development
of the disease. As Nod2 is a pathogen-recognition molecule, it is
therefore of interest to investigate whether Nod2 mutants found in
Crohn's disease patients display an altered ability to detect
bacterial products. Indeed, previous studies had suggested that Nod2
variants found in Crohn's disease displayed an altered sensitivity
toward LPS detection (2). Our findings that Nod2 is a general sensor of
PGNs through the detection of MDP prompted us to investigate whether
PGN or MDP sensing was altered in a Crohn's disease-associated Nod2
mutant. To this end, we compared the ability of wild-type Nod2 and Nod2 3020InsC (Nod2fs), the most frequent Nod2 variant found associated with
Crohn's disease patients, to detect PGNs from Gram-negative or
Gram-positive bacteria (Fig.
3a). Strikingly, while Nod2fs could activate the NF- Innate immunity to microbial pathogens relies on the specific
detection of pathogen-associated molecular patterns (PAMPs) by specific
host receptors (3). A major difficulty concerning the identification of
which PAMP is detected by a specific pathogen-recognition molecule
resides in the fact that most of these products need to be purified
from bacterial cell walls, and therefore contamination with other cell
wall components can often occur and may lead to erroneous conclusions.
As our aim was to investigate the sensing specificity of Nod2 toward
PGNs, we adapted the most stringent purification protocols for PGN
preparations to obtain the highest purity. Using these purified PGN
preparations, we were able to show that Nod2 is a general sensor of
both Gram-negative and Gram-positive PGN. Previous studies showed that
Nod2 displayed varying sensitivities toward different commercial LPSs
(2, 6), which, in light of our data, suggests that these preparations
could be contaminated more or less with PGN, and this possibility
remains to be addressed.
Over the last decades, muramyl peptides and other synthetic derivatives
of this molecule have been reported to be potent immunoadjuvants that
enhance protective immunity against pathogens and tumors by stimulating
immune-competent cells, such as monocytes and macrophages (19, 23).
Despite a long standing investigation in the field of muramyl peptides,
little is known about their interaction with target cells and their
mode of action. Indeed, the nature of the MDP-sensing molecule(s) has
been a subject of controversy (23-25). Interestingly, it has been also
reported that MDP binding sites are located within the intracellular
compartment (26). Our results have defined Nod2 as an intracellular
sensor of MDP, which is in agreement with many of the known biological
properties of MDP. First, Nod2 expression is restricted to
monocytes/macrophages in basal conditions, which is the major cell
population stimulated by MDP. Second, the fact that the MDP sensor is
actually intracellular correlates with the characterization of
cytoplasmic MDP binding sites (26) and would explain why extracellular
presentation of MDP in the absence of lipidic emulsifying compounds
such as liposomes or paraffin oil (the base of Freund's preparation)
is poorly immunostimulatory (27). Indeed, it can now be hypothesized that lipidic moieties might be required to allow MDP to cross the
hydrophobic plasma membrane.
The homeostasis of the intestine relies on a finely tuned surveillance
system mediated by mononuclear cells, including monocytes/macrophages and dendritic cells that are permanent residents of the intestinal mucosa. The constant presence of luminal antigens maintains the tissue
in a state of physiological inflammation through a tightly controlled
equilibrium that balances pro- versus anti-inflammatory signals. Indeed, the discovery that Nod2-dependent
activation of the NF-B pathway following
intracellular stimulation by bacterial products. Recently, mutations in
Nod2 have been shown to be associated with Crohn's
disease, suggesting a role for bacteria-host interactions in the
etiology of this disorder. We show here that Nod2 is a general sensor
of peptidoglycan through the recognition of muramyl dipeptide (MDP),
the minimal bioactive peptidoglycan motif common to all bacteria.
Moreover, the 3020insC frameshift mutation, the most frequent Nod2
variant associated with Crohn's disease patients, fully abrogates
Nod2-dependent detection of peptidoglycan and MDP.
Together, these results impact on the understanding of Crohn's disease
development. Additionally, the characterization of Nod2 as the first
pathogen-recognition molecule that detects MDP will help to unravel the
well known biological activities of this immunomodulatory compound.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B
pathway (2, 5, 6), while others like Nalp1 and Nalp3/cryopyrin have no
known ligands but are also implicated in inflammation (7, 8). The
critical importance of the Nods in inflammatory processes is further
reinforced by the recent characterization of Nod2 and
Nalp3 as susceptibility genes for five hereditary
inflammatory disorders (1, 2, 8-10).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amylase to remove any glycogen and with
trypsin (3× crystallized trypsin, Worthington) digestions to remove
covalently bound proteins (LPXTG proteins in Gram-positive
bacteria) or lipoproteins (Gram-negative bacteria). Samples were
further boiled in 1% SDS to inactivate trypsin and were washed to
remove SDS. Gram-positive samples were treated with 49% hydrofluoridic
acid during 48 h at 4 °C. This mild acid hydrolysis allows
removal of secondary polysaccharides covalently bound to the PGN by
phosphodiester bonds such as teichoic acid, capsules,
poly(
,1-6-GlcNAc), etc. Further treatment of both Gram-positive and
Gram-negative PGNs included washes with 8 M LiCl, 0.1 M EDTA to remove any polypeptidic contamination and with
acetone to remove lipoteichoic acids or any traces of LPS. Samples were
lyophilized to measure PGN amounts. Purity of samples was assessed by
HPLC amino acid and saccharide analysis after HCl hydrolysis (14).
was from R & D Systems.
Zap ExpressTM/EcoRI
vector, Stratagene, catalog number 938202), kindly provided by S. Gisselbrecht (Paris), was screened for Nod2-expressing clones (GenBankTM/EBI accession number CAC42117). A
full-length Nod2-expressing vector was verified by complete automatic
sequencing and inserted into pBKCMV (Stratagene). The Nod2 3020InsC
frameshift mutation was subsequently introduced using QuikChange XL
site-Directed mutagenesis kit (Stratagene). The entire coding region
was verified by sequencing and the size of the encoded product was
verified by immunoblotting. Transfections were carried out in HEK293T
cells as described previously (5).
B Activation Assays--
Studies examining the synergistic
activation of NF-
B by peptidoglycans or MDPs in cells overexpressing
Nod2 were carried out as described by Inohara et al. (6).
Briefly, HEK293T cells were transfected overnight with 30 ng of Nod2
plus 75 ng of Ig
luciferase reporter plasmid. At the same time, 1 µg of PGN preparations or MDPs were added, and the synergistic
NF-
B-dependent luciferase activation was then measured
following 24 h of co-incubation. NF-
B-dependent
luciferase assays were performed in duplicate, and data represent at
least three independent experiments. Data show mean ± S.E.
B activation was assessed by
nuclear translocation of NF-
B p65 in HeLa cells and RAW macrophages
following microinjection of MDPs as described previously (16). At least
50 microinjected cells were examined per coverslip, and experiments
were performed at least two times independently with similar results.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B pathway was measured using a NF-
B-driven luciferase reporter
gene assay. As demonstrated previously (6), transfection of low amounts
(30 ng) of Nod2 itself results in a moderate action of NF-
B
(~5-fold over vector-expressing cells). We observed that PGN
preparations from Gram-negative and Gram-positive bacteria were
similarly efficient in potentiating the Nod2-dependent activation of the NF-
B pathway (Fig.
1a), suggesting that Nod2 is a
general sensor of PGNs. To completely eliminate the possibility that
trace amounts of contaminants in the PGN preparations could be
responsible for some of the Nod2-dependent activation of
NF-
B, PGNs from E. coli and S. aureus were
subjected to additional incubation with polymyxin B or proteinase K and
boiling. These treatments did not lead to any change in the
Nod2-dependent stimulation of the NF-
B pathway compared
with the non-treated PGNs (Fig. 1b).
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Fig. 1.
Nod2 is a general sensor of PGNs through MDP
detection. a, HEK293T cells were transfected with low
amounts of Nod2 expression vector (30 ng) together with 1 µg of each
purified PGN (E.c, E. coli; S.f,
S. flexneri; B.s, B. subtilis;
S.a, S. aureus), and NF- B activity was
measured after 4 h using an NF-
B-luciferase reporter assay, and
fold activation over vector-expressing cells is shown. CTR,
no PGN added. b, PGNs from Gram-negative and Gram-positive
bacteria were subjected to polymyxin B or proteinase K/boiling
treatments to exclude a role for trace amounts of LPS or lipoproteins
in PGN-dependent stimulation of Nod2. NF-
B activity was
measured as in a. Values represent the fold synergy in
luciferase activity compared with activation induced by Nod2 alone.
CTR, buffer minus or plus PGN. c, schematic
representation of PGN repetitive structure, showing that MDP is the
minimal signature of Gram-negative and Gram-positive bacterial PGNs.
d, HEK293T cells were transfected with low amounts of Nod2
(30 ng), Nod1 (30 ng), or TLR2 (50 ng) expression vectors together with
1 µg of either MDP (MDP LD) or the biologically inactive MDP LL, and
NF-
B activity was measured after 4 h using an
NF-
B-luciferase reporter assay. CTR, no MDPs added.
-1,4 linked to N-acetylmuramic acid (MurNAc).
Additionally, the MurNAc residues are substituted with short
DL-amino acid peptides (Fig. 1c). Since GlcNAc
is a sugar moiety also found in LPS, LTA, TA, and other complex
polysaccharides, this sugar is unlikely to account for specific PGN
sensing by Nod2. On the contrary, the MurNAc substituted with two amino
acids, known as muramyl dipeptide (MDP LD or simply MDP), corresponds
to the minimal PGN signature found in all bacteria. We took advantage
of the availability of chemically synthesized MDP, thus avoiding the
risk of contamination by other bacterial products. We observed that
addition of MDP could strongly enhance Nod2-dependent
activation of the NF-
B pathway (Fig. 1d). To test the
specificity of the synergistic activation of Nod2 by MDP, we tested
whether Nod1, another Nod molecule closely related to Nod2, could also
sense MDP. Strikingly, Nod1-dependent activation of the
NF-
B pathway was completely insensitive to MDP (Fig. 1d).
Since TLR2 has been shown to be implicated in PGN detection (18), we
investigated whether it could sense MDP. Extracellular stimulation of
TLR2-transfected cells with MDP (1 µg) failed to potentiate the
TLR2-dependent activation of the NF-
B pathway (Fig.
1d), while addition of commercial preparations of S. aureus PGN could do so (data not shown). This result suggests
either that TLR2 might require other yet unknown structural PGN motifs
to achieve recognition, or, alternatively, that co-receptors would be
required for this recognition. Therefore, Nod2 is the only
pathogen-recognition molecule identified so far that detects PGN
through MDP, its minimal invariant motif.
B
pathway by MDP LL (Fig. 1d). Even though we cannot exclude
that pathogen-recognition molecules distinct from Nod2 could also
detect MDP, these observations strongly suggest that Nod2 plays a key
role in mediating the biological activities of MDP.
B pathway was assessed by immunostaining
through the detection of nuclear translocation of the NF-
B p65
subunit in activated cells. Epithelial cells and macrophages were first
stimulated by extracellular addition of MDP LD or MDP LL. In neither
case could we observe any activation of the NF-
B pathway (Fig.
2a). As positive controls, epithelial cells and macrophages were stimulated by TNF
and LPS, respectively. Therefore, these results suggest that none of these cells
possess pathogen-recognition molecules at their plasma membrane that
could detect MDP and trigger downstream signaling pathways. As
macrophages express most of the TLRs, including TLR2 and TLR4, this
observation is consistent with our findings that TLR2 is unable to
detect MDP. Since Nod2 is a cytoplasmic protein, we investigated
whether MDP detection could occur inside microinjected cells.
Epithelial cells and macrophages were then microinjected with MDP LD or
MDP LL together with fluorescein isothiocyanate-dextran, to track
microinjected cells. While neither epithelial cells nor macrophages
were stimulated by intracellular presentation of MDP LL, 80% of MDP
LD-microinjected macrophages displayed activation of the NF-
B
pathway as observed by nuclear translocation of the NF-
B p65 subunit
(Fig. 2b). In contrast, 100% of MDP LD-microinjected epithelial cells remained NF-
B-inactive (Fig. 2b).
Together, these results are in agreement with our observation that
Nod2, which is an intracellular pathogen-recognition molecule expressed in macrophages, detects MDP LD but not MDP LL. Moreover, these observations demonstrate that epithelial cells, which do not express Nod2, do not possess an additional pathogen-recognition molecule distinct from Nod2 that could either extracellularly or intracellularly detect MDP.
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Fig. 2.
Intracellular sensing of MDP in macrophages
but not in epithelial cells. a, extracellular
addition of MDP (MDP LD) or MDP LL in epithelial cells or macrophages
fails to activate the NF- B pathway, as observed in immunostaining by
the lack of p65 NF-
B subunit nuclear translocation. Positive
controls were TNF
(100 ng/ml) and LPS (1 µg/ml) stimulation for
epithelial cells and macrophages, respectively. b,
microinjection of MDP (MDP LD) and MDP LL into macrophages and
epithelial cells. Immunostaining of the p65 subunit of NF-
B reveals
nuclear translocation in macrophages but not in epithelial cells
microinjected with MDP.
B pathway as well as wild-type Nod2 when overexpressed, which is consistent with previous reports (2), the
activation of the NF-
B pathway by Nod2fs was absolutely not potentiated by the addition of any PGN, thus showing that Nod2fs is
unable to detect PGNs. This defect in PGN sensing was further illustrated by the fact that Nod2fs is also deficient in
detecting MDP (Fig. 3b). Therefore, these results suggest
that defects in MDP sensing influence the development of Crohn's
disease in patients with Nod2 mutations.
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Fig. 3.
3020insC, the most frequent Nod2 mutation
associated with Crohn's disease, abrogates Nod2-dependent
sensing of PGNs and MDP. a, HEK293T cells were
transfected with Nod2 (30 ng) or Nod2fs (20 ng) expression vectors
together with 1 µg of each purified PGN (E.c, E. coli; S.f, S. flexneri; B.s,
B. subtilis; S.a, S. aureus), and
NF- B activity was measured after 4 h using an
NF-
B-luciferase reporter assay. CTR, no PGN added.
b, HEK293T cells were transfected with Nod2 (30 ng) or
Nod2fs (20 ng) expression vectors together with 1 µg of either MDP
(MDP LD) or MDP LL, and NF-
B activity was measured after 4 h
using an NF-
B-luciferase reporter assay. CTR, no MDPs
added.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B pro-inflammatory cascade is modulated by PGN
sensing in these cells highlights the essential role of Nod2 in the
maintenance of mucosal homeostasis. In Crohn's disease patients
carrying mutations in Nod2, the manifestation of pathology is then
likely to be related to a defect in mononuclear-driven homeostasis at
the level of the intestinal mucosa. Our discovery of MDP as the
bacterial ligand that initiates Nod2-dependent signaling
and the insensitivity of a Crohn's disease-associated Nod2 mutation to
this bacterial product underscores the critical role of bacterial
sensing by Nod2 in intestinal mucosal homeostasis. Therefore, it will
be of interest to examine whether some of the Nod2-independent Crohn's disease susceptibility alleles are associated with defects in pathways
that are involved in the processing of PGN, such as lysozyme in the
endosomal compartment.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the assistance of Muguette Jéhanno as well as Martine Parmier who helped with plasmid preparations. We also acknowledge Didier Blanot for the amino acid and saccharide analysis of the PGN samples. We thank those individuals that donated bacterial strains and plasmids. We also thank the "TLR Group" at the Institut Pasteur and Bill Goldman for helpful discussion.
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FOOTNOTES |
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* This work was supported in part by a grant from the Institut Pasteur, "Program Transversal de Recherche" (to S. E. G. and D. J. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a grant from Danone Vitapole, Paris, France.
Supported by a postdoctoral fellowship from the
Fundação para a Ciência e a Tecnologia, Portugal
(SFRH/BPD/1567/2000).
§§ Supported by a grant from the Ministère Français de l'Education Nationale.
¶¶ These authors share senior authorship.
To whom correspondence should be addressed. Tel.:
33-1-45-68-89-93; Fax: 33-1-40-61-39-02; E-mail: philpott@pasteur.fr.
*** Howard Hughes International Research Scholar.
Published, JBC Papers in Press, January 13, 2003, DOI 10.1074/jbc.C200651200
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ABBREVIATIONS |
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The abbreviations used are: PGN, peptidoglycan; MDP, muramyl dipeptide; HPLC, high performance liquid chromatography; LPS, lipopolysaccharide; TNF, tumor necrosis factor; LTA, lipoteichoic acid; TA, teichoic acid; PAMP, pathogen-associated molecular pattern.
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