Lipoteichoic Acid Induces Unique Inflammatory
Responses when Compared to Other Toll-Like Receptor 2
Elizabeth M. Long1, Brandie Millen2, Paul Kubes2, Stephen M. Robbins1*
1Departments of Oncology, Biochemistry and Molecular Biology and the Southern Alberta Cancer Research Institute, University of Calgary, Calgary, Canada,
2Department of Biophysics and Physiology and The Institute of Infection, Immunity, and Inflammation, University of Calgary, Calgary, Canada
Toll-like receptors (TLRs) recognize evolutionarily-conserved molecular patterns originating from invading microbes. In this
study, we were interested in determining if microbial ligands, which use distinct TLR2-containing receptor complexes,
represent unique signals to the cell and can thereby stimulate unique cellular responses. Using the TLR2 ligands, R-FSL1, S-
FSL1, Pam2CSK4, Pam3CSK4, and lipoteichoic acid (LTA), we demonstrate that these ligands activate NF-kB and MAP Kinase
pathways with ligand-specific differential kinetics in murine macrophages. Most strikingly, LTA stimulation of these
pathways was substantially delayed when compared with the other TLR2 ligands. These kinetics differences were associated
with a delay in the LTA-induced expression of a subset of genes as compared with another TLR2 ligand, R-FSL1. However,
this did not translate to overall differences in gene expression patterns four hours following stimulation with different TLR2
ligands. We extended this study to evaluate the in vivo responses to distinct TLR2 ligands using a murine model of acute
inflammation, which employs intravital microscopy to monitor leukocyte recruitment into the cremaster muscle. We found
that, although R-FSL1, S-FSL1, Pam2CSK4, and Pam3CSK4 were all able to stimulate robust leukocyte recruitment in vivo,
LTA remained functionally inert in this in vivo model. Therefore distinct TLR2 ligands elicit unique cellular responses, as
evidenced by differences in the kinetic profiles of signaling and gene expression responses in vitro, as well as the
physiologically relevant differences in the in vivo responses to these ligands.
Citation: Long EM, Millen B, Kubes P, Robbins SM (2009) Lipoteichoic Acid Induces Unique Inflammatory Responses when Compared to Other Toll-Like Receptor
2 Ligands. PLoS ONE 4(5): e5601. doi:10.1371/journal.pone.0005601
Editor: Dominik Hartl, LMU University of Munich, Germany
Received February 25, 2009; Accepted April 16, 2009; Published May 19, 2009
Copyright: ? 2009 Long et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by a Canadian Institutes for Health Research Group Grant in Inflammatory Diseases and an operating grant from the Alberta
Cancer Board, SMR holds a Canadian Research Chair and is an AHFMR Research Scientist, P.K. holds a Canadian Research Chair and The Snyder Chair in Critical
Care Medicine and is an AHFMR Research Scientist, EML was supported by studentships from Natural Sciences and Engineering Research Council of Canada and
AHFMR. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Toll-like receptors (TLRs) are key components of the immune
system’s capacity to recognize infectious non-self and to mount a
rapid and effective immune response . They are type I
transmembrane receptors, composed of an extracellular, leucine
rich repeat (LRR), ligand-recognition motif, as well as a highly
conserved, cytoplasmic, Toll/IL-1R (TIR), signaling-initiating
domain [2,3]. These receptors have evolved to recognize microbial
molecules of bacterial, viral, and fungal origin that are comprised
of molecular structures as diverse as proteins, lipopeptides,
glycolipids, as well as nucleic acids. These ligands bind to either
homo- or hetero-dimers of the TLR’s, often in combination with
different co-receptors .
TLR2, forming a heterodimer with either TLR1 or TLR6, is
responsible for the recognition of bacterial lipoproteins and
lipopeptides . These ligands are derived from the bacterial cell
membrane, where they are anchored via lipid chains attached to a
polypeptide at a conserved N-terminal cysteine residue . The
number of fatty acids coupled to the N-terminus of the polypeptide
is the crucial determinant in the ligand preference for specific
TLR2 heterodimers. Triacylated lipoproteins are produced by
most bacteria, with the exception of mycoplasma, and are
recognized by TLR2/1 heterodimer complexes . The third
acyl chain in triacylated lipoproteins is attached via an amide bond
to the N-terminal cysteine. This reaction depends on the presence
of an N-acetyltransferase that is absent in mycoplasma and
therefore these organisms produce only diacylated lipopeptides,
which are recognized by TLR2/6 heterodimer complexes [8,9]. In
addition to these lipoproteins, TLR2 also recognizes and responds
to the Gram-positive bacterial cell wall component, lipoteichoic
acid (LTA) . LTA is a diacylated, glycerophosphate polymer
and as such, this ligand is recognized by a TLR2/6 heterodimer
complex, presumably in a similar manner as the diacylated
lipoproteins . In short, the TLR2 heterodimer complexes
allow for the accommodation of a structurally diverse ligand
repertoire. We were particularly interested in whether the use of
these distinct heterodimer complexes would translate to distinct,
receptor complex-specific, responses to TLR2 ligands.
TLR1, TLR2, and TLR6 are all members of the TLR1-family
of TLRs. These receptors share 66% sequence identity and are
located in tandem on the same chromosome in mammals .
The sequence similarity between TLR1 and TLR6 is in part the
result of gene conversion in a region encompassing the last four
PLoS ONE | www.plosone.org1 May 2009 | Volume 4 | Issue 5 | e5601
LRR motifs, the C-terminal cap, the transmembrane domain, and
three quarters of the TIR domain . These boundaries of gene
conversion are tightly conserved across species, presumably as a
result of an evolutionary pressure imparted by TLR1 and TLR6
shared functions, such as dimerization with TLR2 and the
initiation of signaling responses. Despite the strict similarities
between TLR1 and TLR6 there are some areas of divergence
within both the LRR-motifs and the TIR domains. We
hypothesized that the areas of free divergence within the TIR
domain and at the C-terminus of the proteins may have evolved
unique structure-function relationships, which could thereby
impart ligand-specific cellular responses to TLR2-containing
receptor complexes. Interestingly, employing distinct heterodimers
is not the only way that TLR2 is able to extend its ligand
repertoire, a forward genetics screen carried out by Beutler and
colleagues, has revealed the requirement for the co-receptor,
CD36, in order to mount a productive response to both LTA and
the R-enantiomer of diacylated lipopeptides .
Based on the divergent sequences within the TLR2 binding
partners TLR1 and TLR6, and the use of distinct, ligand-specific
co-receptors, we questioned whether these differences would
impart ligand-specific cellular responses to distinct TLR2 ligands.
We evaluated the response of both immortalized and primary
macrophages to different ligands that have been shown to signal
through TLR2/1, TLR2/6, or TLR2/6/CD36. The TLR ligands
used in this study were: the triacylated peptide, Pam3CSK3
(TLR2/1), the diacylated peptides, Pam2CSK4 (TLR2/6), S-
FSL1 (TLR2/6), R-FSL1 (TLR2/6/CD36), and the diacylated
glycerophosphate polymer, LTA (TLR2/6/CD36). We found that
the different TLR2 ligands stimulated downstream signaling
pathways with ligand-specific kinetic profiles in murine macro-
phages. Most strikingly, LTA consistently activated these pathways
with a substantially delayed kinetic profile as compared with the
other ligands. In addition, there was a delay in the rate of
production of a subset of TLR-responsive transcripts in response
to LTA (TLR2/6/CD36), as compared with R-FSL1 (TLR2/6/
CD36). However, 4 hours following the initial ligand administra-
tion, the transcriptional profiles activated by each ligand were
equivalent. Upon evaluating the in vivo pro-inflammatory
potential of these ligands, we found that although Pam3CSK3
(TLR2/1), Pam2CSK4 (TLR2/6), S-FSL1 (TLR2/6), and R-
FSL1 (TLR2/6/CD36), each induced robust leukocyte recruit-
ment into the cremaster muscle, LTA (TLR2/6/CD36), was
unable to cause any significant leukocyte recruitment. Therefore
we have shown that there are differences in the interpretation of
signals derived from ligands that signal through distinct TLR2-
containing receptor complexes, as evaluated using both in vitro
and in vivo assays.
Materials and Methods
Antibodies and Reagent
Pam3CSK4 and Pam2CSK4 were purchased from InvivoGen
(San Diego, CA). R- and S-FSL1 were purchased from EMC
microcollections (Tubingen, Germany). Ultrapure LTA was
provided by Dr. T. Hartung (University of Konstanz, Konstanz,
Germany). In this study we chose to use only synthesized di- or
triacylated ligands as well as highly purified LTA, in order to avoid
any of the prevailing issues of contamination. Antibodies for
phospho-SAPK/JNK, total-SAPK/JNK, phospho-erk1/2, total-
erk1/2, phospo-p38, phospho-c-jun, total-c-jun, phospho-ATF2,
and IkBa were purchased from Cell Signaling Technologies
(Beverly, MA). Anti-total p38 antibody was purchased from Santa
Cruz Biotechnology (Santa Cruz, CA).
Cell Culture and Western blotting
Western blots were performed as previously described .
Briefly, Raw 264.7 cells were plated in six-well tissue culture plates
and allowed to grow to 85% confluence. The cells were treated
with ligand for the indicated times, then lysed in Laemmili sample
buffer and sonicated. The samples were then resolved using 10%
SDS-PAGE. After electrophoresis, proteins were transferred to
nitrocellulose membranes, which were then blocked by incubation
in 5% BSA in TBS plus 0.05% Tween 20 (TBST). The
membranes were then incubated at 4uC overnight in the primary
antibody, washed for 40 minute in TBST, incubated for 1 h in
secondary antibody, and washed for another 40 minute in TBST.
The membranes were developed using an ECL substrate. For
experiments using primary bone marrow derived macrophages,
bone marrow was isolated from the femoral, tibial, and pelvic
bones of wildtype C57BL/6 or TLR2-/- mice. These cells were
cultured in DMEM + 10% FBS + penicillin/streptomycin/
glutamine + 10% L929 cell conditioned media for 7 days, feeding
the cells with fresh media on day 5. Treatments and Western blots
were then performed as described above. Image J software (NIH)
was used to quantify the Western blot signals.
RNA was isolate from relevant cells using TrizolH (Invitrogen)
according to the manufacturer’s protocol. Total RNA was treated
with DNAase I and further purified by phenol extraction and
precipitated with sodium acetate and ethanol. cDNA was prepared
using superscript II (Invitrogen). For semi-quantitative RT-PCR the
cDNA was amplified using the following gene-specific primers for
TNFa, 59TTGACCTCAGCGGCTGAGTTG39 and 59CCTGT-
AGCCCACGTCGTAGC39. The PCR products were prepared
with 106 DNA loading dye and run on a 2% TAE-agarose gel.
saturated pixels function engaged so as to avoid overexposing the
image. Image J software (NIH)was used to quantifiy the bands on the
gel. In the case of the SABiosciences arrays, cDNA was diluted with
water according to the manufacture’s instructions and further mixed
with the PCR mastermix supplied by the manufacture. 100 mL of this
final mixture was added to each well of the array, (RT2Profiler PCR
Array, Mouse Inflammatory Cytokines and Receptors), and run in
the ABI7000 RT-PCR machine using the following protocol, 95uC
10 minutes, 40 cycles of 15 seconds 95uC, and 1 minute 60uC. The
normalized threshold cycle (Ct) value for each gene, (DCt), was
determined by subtracting the Ctvalue of the housekeeping gene,
actin, from the Ctvalue of the gene of interest. The change in the Ct
value between control and treated samples for each gene of interest,
(DDCt), was then calculated and the fold change was determined
using the following formula, 2‘(2DDCt).
Experiments were performed using male C57/B6 wildtype mice
purchased from The Jackson Laboratory (Bar Harbor, ME) or
TLR2-/- provided by Prof. Shizuo Akira (Osaka University,
Japan). These mice were maintained in a pathogen-free facility at
the University of Calgary’s Animal Resource Center. At the time
of use, mice weighed between 20 and 30 g and were 6–10 wk old.
Experimental animal protocols performed in this study were
approved by the University of Calgary Animal Care Committee
and met the guidelines of the Canadian Council for Animal Care.
In vivo evaluation of TLR2 ligands
Wildtype C57/B6 mice were given an intrascrotal injection of
150 mL of saline alone, or saline containing, TNFa (20 ng/g), LPS
Differential TLR2 Responses
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The authors thank Prof. S. Akira for kindly providing the TLR2-/- mice;
Dr. Alexander C. Klimowicz and Maja B. Hoegg for a helpful discussion
and review of the manuscript; and L. Zbytnuik and D. Brown for their
expert assistance in animal care.
Conceived and designed the experiments: EML. Performed the experi-
ments: EML BM. Analyzed the data: EML SMR. Contributed reagents/
materials/analysis tools: PK SMR. Wrote the paper: EML.
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