INFECTION AND IMMUNITY, Dec. 2006, p. 6829–6838
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 74, No. 12
Toll-Like Receptors Differentially Regulate CC and CXC Chemokines
in Skeletal Muscle via NF-?B and Calcineurin?
John H. Boyd,1,2Maziar Divangahi,1Linda Yahiaoui,1Dusanka Gvozdic,1
Salman Qureshi,3and Basil J. Petrof1,2*
Meakins-Christie Laboratories, McGill University, Montreal, Canada1; Respiratory Division, McGill University Health Center,
Montreal, Canada2; and Critical Care Division, McGill University Health Center, Montreal, Canada3
Received 21 February 2006/Returned for modification 5 April 2006/Accepted 6 September 2006
Immunologically active molecules such as cytokines and chemokines have been implicated in skeletal muscle
weakness during sepsis as well as recovery from muscle injury. In sepsis, Toll-like receptors (TLRs) act as key
sentinel molecules of the innate immune system. Here we determined skeletal muscle cell responses of two
prototypical CC and CXC chemokine genes (monocyte chemoattractant protein 1 [MCP-1] and KC, respec-
tively), to stimulation with specific TLR ligands. In addition, we examined whether NF-?B and calcineurin
signaling are involved in these responses. Differentiated myotubes and intact whole muscles expressed TLR2,
TLR4, TLR5, and TLR9. Stimulation with ligands for TLR2 (peptidoglycan) or TLR4 (LPS) elicited robust and
equivalent levels of MCP-1 and KC mRNA expression, whereas stimulation of TLR5 (by flagellin) required
gamma interferon priming to induce similar effects. Although both TLR2 and TLR4 ligands activated the
NF-?B pathway, NF-?B reporter activity was approximately 20-fold greater after TLR4 stimulation than after
TLR2 stimulation. Inhibitory effects of NF-?B blockade on TLR-mediated chemokine gene expression, by
either pharmacological (pyrrolidine dithiocarbamate) or molecular (IKK? dominant-negative transfection)
methods, were also more pronounced during TLR4 stimulation. In contrast, inhibitory effects on TLR-
mediated chemokine expression of calcineurin blockade (by FK506) were greater for TLR2 than for TLR4
stimulation. MCP-1 and KC mRNA levels also demonstrated differential responses to NF-?B and calcineurin
blockade during stimulation with specific TLR ligands. We conclude that skeletal muscle cells differentially
utilize the NF-?B and calcineurin pathways in a TLR-specific manner to enable complex regulation of CC and
CXC chemokine gene expression.
Chemokines consist of a large family of low-molecular-
weight cytokines which are involved in directing the recruit-
ment and activation of leukocytes to sites of infection or in-
flammation (34). The chemokines have been broadly divided
into CC, CXC, C, and CX3C subgroups, based upon the po-
sitioning of amino acids relative to the first two conserved
cysteine residues (34). In general, CC chemokines act predom-
inantly upon monocytes/macrophages, eosinophils, basophils,
and lymphocytes, whereas CXC chemokines are able to attract
neutrophils (34). Increased expression of multiple chemokines
has been implicated in the infiltration of skeletal muscle by
macrophages and lymphocytes in muscular dystrophies (11, 33)
and inflammatory myopathies (10, 45). Along these same lines,
we have reported increased chemokine expression in the mu-
rine diaphragm during acute sepsis (12) as well as in mice
suffering from muscular dystrophy (11), two conditions which
are associated with increased leukocyte trafficking to this vitally
important muscle (5, 11, 29, 42). In addition, chemokines ap-
pear to play an important function in skeletal muscle repair
(49, 50) and could thus play a key role in facilitating recovery
from various forms of muscle fiber injury, including that in-
duced by sepsis (14, 29).
The early host response to sepsis is mediated by the innate
immune system, through identification of foreign pathogens
and initiation of a proinflammatory cascade. The capacity to
mount a vigorous host immune response without prior expo-
sure to a particular pathogen is mediated by a family of Toll-
like receptors (TLRs) that recognize unique microbial struc-
tures termed pathogen-associated molecular patterns (43, 48).
The TLR family includes more than 10 members, with different
ligand specificities and differential expression among cell types
(32, 54). TLRs are type I transmembrane receptors containing
an extracellular leucine-rich repeat domain linked to a cyto-
plasmic Toll/interleukin-1 receptor homology domain. Activa-
tion of TLRs generally leads to nuclear translocation of the
transcription factor NF-?B, a critical component within many
proinflammatory pathways (21), including those associated
with chemokine gene expression (6, 15). Although TLRs share
the ability to activate NF-?B (43), cross talk with other signal-
ing pathways is one mechanism by which activation of specific
TLRs may lead to different patterns of gene expression. An-
other key signaling molecule of the immune response, the
calcium/calmodulin-dependent phosphatase known as cal-
cineurin, has been reported to have a functional interaction
with NF-?B (46). Interestingly, calcineurin also plays an im-
portant role in the growth, differentiation, and specialization of
skeletal muscle (9, 31, 40).
The majority of previous studies investigating the immuno-
logical attributes of skeletal muscle have been directed at elu-
cidating its ability to participate in the adaptive immune re-
sponse. For example, muscle fibers of patients suffering from
inflammatory myopathies and primary skeletal muscle cell cul-
* Corresponding author. Mailing address: Respiratory Division,
Room L411, Royal Victoria Hospital, 687 Pine Ave. West, Montreal,
Quebec H3A 1A1, Canada. Phone: (514) 934-1934, ext. 35946. Fax:
(514) 843-1695. E-mail: firstname.lastname@example.org.
?Published ahead of print on 18 September 2006.
tures exposed to proinflammatory stimuli express human leu-
kocyte antigen class I/II and costimulatory molecules (52).
These are involved in the priming and activation of lympho-
cytes, and coculture experiments have confirmed that antigen-
exposed skeletal muscle cells are capable of inducing lympho-
cyte activation (53). The chemokines interleukin-8 (IL-8)
(CXCL8), RANTES (CCL5), and monocyte chemoattractant
protein 1 (MCP-1) (CCL2) are produced by human myoblasts
under proinflammatory conditions, and it has been speculated
that these molecules may be important in autoimmune muscle
disease (13). However, the extent to which skeletal muscle cells
are capable of participating in the innate immune response is
less well understood. It should be noted that innate immunity
is not only involved in combating infection, but may also play
a more general role in sensing “danger signals” related to other
types of cellular injury or stress (48).
TLRs are the main sentinel molecules responsible for trig-
gering the innate immune response and may therefore consti-
tute an important pathway for inducing chemokine expression
by skeletal muscle cells. Accordingly, the principal aim of our
study was to evaluate the role of TLR-mediated signaling in
the process of chemokine gene expression by skeletal muscle.
We selected MCP-1 (CCL2) and KC (CXCL1) as prototypical
members of the two largest chemokine families (CC and CXC,
respectively). These two chemokine families have been impli-
cated in different disease states and have also been reported to
show differential regulation under various pathological condi-
tions (10, 30). Our specific objectives in this study were as
follows: (i) to ascertain the skeletal muscle cell expression
pattern of different TLRs known to be active in bacterial or
viral recognition, (ii) to determine whether there is differential
regulation of MCP-1 and KC gene expression after stimulation
of these TLRs by their respective ligands, and (iii) to evaluate
the involvement of NF-?B and calcineurin in the regulation of
MCP-1 and KC gene expression after specific TLR stimulation
in skeletal muscle cells.
MATERIALS AND METHODS
Cell culture and functional stimulation with TLR ligands. Myoblasts from the
murine skeletal muscle cell line C2C12 (American Type Culture Collection,
Manassas, VA) were grown on six-well plates coated with Matrigel (1 mg/ml in
Dulbecco’s modified Eagle’s medium [DMEM]; Becton-Dickinson, Franklin
Lakes, NJ). The cells (passage 3 or below) were expanded in growth medium
consisting of DMEM with 10% fetal bovine serum. After reaching approximately
70% confluence, the cells were switched to differentiation medium composed of
DMEM with 2% horse serum to induce myoblast fusion into differentiated
myotubes. On day 5 in differentiation medium, myotubes were incubated with
one of the following TLR ligands (43) diluted in DMEM with 5% heat-inacti-
vated fetal bovine serum for a period of 4 h: 10 ?g/ml Staphylococcus aureus
peptidoglycan (PGN) (for TLR2), 25 ?g/ml poly(I:C) (for TLR3), 1 ?g/ml
Escherichia coli O111:B4 ultrapure lipopolysaccharide (LPS) (for TLR4), 1
?g/ml Salmonella enterica serovar Typhimurium flagellin (for TLR5), 1 mM
loxoribine (for TLR7), and 1 ?M unmethylated CpG DNA consisting of phos-
(for TLR9) (37), where underlining indicates CpG sequences. TLR2, TLR3,
TLR4, TLR5, and TLR7 ligands were purchased from Invivogen (San Diego,
California); TLR9 ligand was obtained from Alpha DNA (Montreal, Quebec,
Canada). Primary skeletal muscle cell cultures were derived from C57BL/6 mice
and TLR2 null mutants on the same background strain (44), using single living
muscle fibers isolated from the tibialis anterior limb muscle, according to pro-
cedures which we have previously described in detail (11). The primary cultures
were stimulated on the fifth day after induction of differentiation, with either
PGN or LPS at the same doses mentioned above.
Pharmacological and dominant-negative inhibition studies. Treatment with
pyrrolidine dithiocarbamate (PDTC; Fisher Scientific, Nepean, Ontario, Can-
ada), which acts an inhibitor of NF-?B by blocking the E3 ligase responsible for
I?B degradation (20), was initiated 24 h before and maintained during the 4-h
TLR stimulation period at a dose of 100 ?M (27). Treatment with FK506 (Eton
Bioscience, Inc., San Diego, California), an inhibitor of calcineurin, was similarly
applied for 24 h before and during TLR stimulation, at a dose of 100 ng/ml (40).
To evaluate the role of free radical species, cells were treated with one of two
antioxidants, N-acetylcysteine (10 mM) or catalase (2,000 U/ml) (Fisher Scien-
tific), initiated 1 h before and maintained during TLR stimulation; these doses
have previously been shown to block reactive oxygen species-mediated IL-6
upregulation in skeletal muscle cells (25). All of the above studies were per-
formed on C2C12 myotubes after 5 days in differentiation medium as described
A dominant-negative mutant form of the I?B kinase, IKK? (provided by P.
Barker, McGill University, Montreal, Canada), was employed to inhibit NF-?B
activation as previously described (3). Myoblasts transfected with a vector con-
taining the same plasmid backbone (pAdtrack) served as controls. C2C12 cells
(5 ? 105) were seeded onto 60-mm plates, incubated overnight in growth me-
dium, and transfected the following day at approximately 50% confluence. To
achieve transfection, Lipofectamine 2000 (Invitrogen, Carlsbad, CA) and 8 ?g of
plasmid DNA were combined according to the manufacturer’s protocol and
placed onto the monolayer of cells in the presence of Optimum medium (In-
vitrogen) for 4 h. The transfection mixture was subsequently replaced with
growth medium for 24 h. The cells were then replated in Matrigel-coated six-well
plates and switched to differentiation medium. Stimulation experiments with
TLR ligands were performed 5 days later as described earlier.
Evaluation of NF-?B pathway activation. To assess I?B? phosphorylation and
degradation after TLR stimulation, myotubes were harvested from six-well plates
and placed in cell lysis buffer (Cell Signaling, Danvers, MA) containing aprotinin
(10 ?g/ml), leupeptin (10 ?g/ml), and the phosphatase inhibitor sodium or-
thovanadate (10 mM). Total protein concentration was determined by Bradford
assay (Bio-Rad, Hercules, CA). Protein samples were size separated on a 10%
sodium dodecyl sulfate–polyacrylamide gel and transferred onto polyvinylidene
difluoride membranes. Immunodetection was performed with primary antibodies
directed against total I?B? and phosphorylated I?B? (both from Cell Signaling),
which were reacted with membranes blotted with 20 ?g and 60 ?g protein per
lane, respectively. The immunoblot signals were revealed using appropriate
horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse secondary
antibodies, in conjunction with the ECL detection system (Amersham, Piscat-
NF-?B transcriptional activity was evaluated using a NF-?B response element-
driven firefly luciferase reporter plasmid (pNF-?B-Luc; Clontech, Mountain
View, CA), which was transfected into C2C12 cells prior to TLR stimulation.
The pNF-?B-Luc vector was cotransfected into C2C12 myoblasts together with
a thymidine kinase promoter-driven Renilla luciferase vector (pRL-TK; Pro-
mega, Madison, WI), which is constitutively active at a low level. For each 60-mm
plate, 7.7 ?g of pNF-?B-Luc and 0.3 ?g of pRL-TK were used in conjunction
with Lipofectamine as described earlier. The Dual-Luciferase reporter assay
system (Promega) was used to quantify the activity of both luciferase reporters
within the same samples, according to the manufacturer’s instructions. Light
emission was measured in an Lmax 384 luminometer (Molecular Devices, Down-
ingtown, PA). All results were expressed as the ratio of NF-?B firefly luciferase
to Renilla luciferase activity in relative light units, using the constitutively active
Renilla as an internal control to adjust for differences in transfection efficiency.
Analysis of MCP-1 and KC gene expression. RNase protection assays were
employed to quantify MCP-1 and KC mRNA levels. Myotubes were harvested at
4 h after stimulation with the various TLR ligands, in the presence or absence of
NF-?B/calcineurin inhibition as described above, and total RNA was extracted.
32P-labeled riboprobes were synthesized using a custom-made mouse multiprobe
kit (BD Biosciences). The riboprobes were hybridized with each RNA sample
(10 ?g) overnight at 56°C according to the manufacturer’s instructions. The
protected RNA fragments were separated using a 5% polyacrylamide gel and
detected by autoradiography. Bands representing the individual mRNA species
were then quantified using an image analysis system (FluorChem 8000; Alpha
Innotech Corp, San Leandro, CA), and the signals were normalized to the L32
housekeeping gene to control for any loading differences across lanes as previ-
ously described (16, 17).
Reverse transcription (RT)-PCR detection of TLR expression. Intact dia-
phragm and tibialis anterior muscles of C57BL/10 mice (8 to 10 weeks of age)
were snap-frozen in liquid nitrogen immediately after euthanasia. Primary myo-
tubes from the diaphragm and tibialis anterior were derived from dissociated
single fibers as previously described in detail (11). Myoblasts and myotubes from
6830 BOYD ET AL.INFECT. IMMUN.
C2C12 cells were prepared as described above. Total RNA from muscle tissues
and cell cultures was extracted using TRIzol (Invitrogen) according to the man-
ufacturer’s instructions. The RNA (1 ?g) was treated with DNase I (Invitrogen)
and reverse transcribed using Moloney murine leukemia virus reverse transcrip-
tase and random primers. Negative controls lacking reverse transcriptase were
included in all cases to exclude genomic DNA contamination. PCR amplification
was performed for 35 cycles at 94°C for 45 s, 55°C for 45 s, and 72°C for 45 s. The
following primers were used (5? to 3?): TLR2 Forward, CACCATTTCCACGG
ACTGTGGTACCTG; TLR2 Reverse, CTGGCCTTATCCAAGGGCCACTC
CAG; TLR3 Forward, GACTGGGTCTGGGAACATTTCTCC; TLR3 Reverse,
AGCTCCATGTGCTACTTGCAATTTGT; TLR4 Forward, ATCTACTCGAG
TCAGAATGAGGACTGG; TLR4 Reverse, CTCTGCTGTTTGCTCAGGAT
TCGAGGC; TLR5 Forward, GGAATCTGTTTCCTGTGTGCTATAAGACC;
TLR5 Reverse, ATGGTTGCTATGGTTCGCAACTGGATGG; TLR7 For-
ward, TCTATTCAGAGGCTCCTGGATGAC; TLR7 Reverse, CTTCAGGTA
CCAAGGGATGTCCTA; TLR9 Forward, CTAGACGTGAGAAGCAACCCT
CTG; TLR9 Reverse, CAGCTCGTTATACACCCAGTCGGC; HPRT Forward,
GTGGATACAGGCCAGACTTTGTTG; HPRT Reverse, GAGGGTAGGCTG
GCCTATTGGCT. All PCR products were electrophoresed on 1% agarose gels
containing ethidium bromide and visualized under UV light.
Statistical analysis. All data are expressed as means ? standard errors (SE).
For each experimental condition and time point, four independent replicate
analyses were performed, unless otherwise noted. Groups were compared
using the Mann-Whitney rank sum test. The analyses were performed using
SigmaStat software (SPSS, Chicago, IL), and statistical significance was set at
a P value of ?0.05.
TLR expression patterns in cultured muscle cells and whole
muscles. TLR2, TLR4, and TLR5 are primarily involved in the
recognition of bacterial surface structures, whereas TLR3,
TLR7, and TLR9 recognize nucleic acids (43). RT-PCR was
employed to determine whether these TLRs are expressed in
skeletal muscle cells, using C2C12 cells and primary (from
diaphragm and tibialis anterior) skeletal muscle cell cultures.
In addition, whole-tissue samples from diaphragm and tibialis
anterior muscles obtained in vivo were examined in the same
manner. As shown in Fig. 1 (upper panel), TLR2, TLR4,
TLR5, and TLR9 were expressed in whole muscles obtained in
vivo as well as in differentiated myotubes from C2C12 or pri-
mary cultures. On the other hand, we were unable to detect
TLR3 or TLR7 under the same experimental conditions (Fig.
1, lower panel). Interestingly, TLR2 and TLR5 were expressed
in C2C12 myotubes but not myoblasts, indicating that the ex-
pression pattern of these TLRs is regulated by the state of
skeletal muscle cell differentiation.
Functional responses to stimulation of individual TLRs. As
shown in Fig. 2, an RNase protection assay was used to eval-
uate MCP-1 and KC gene expression at the mRNA level in
C2C12 myotubes stimulated with specific TLR ligands. Con-
sistent with their lack of detectable expression by RT-PCR,
there was no response to TLR3 ligand [poly(I:C)] or TLR7
ligand (loxoribine) stimulation. In contrast, there was marked
induction of MCP-1 and KC expression after 4 h of incubation
with TLR2 ligand (PGN) or TLR4 ligand (LPS). Interestingly,
although TLR5 and TLR9 expression were observed by RT-
PCR as shown in Fig. 1, stimulation with their respective li-
gands failed to elicit detectable expression of MCP-1 or KC
mRNA (Fig. 2).
We next examined whether TLR5 or TLR9 responses could
be induced by initially priming myotubes with gamma inter-
feron (IFN-?), since this has been shown to increase TLR
expression as well as responsiveness in other systems (19, 47).
IFN-? priming for 24 h in the absence of TLR ligand stimula-
tion resulted in a small but detectable upregulation of KC and
MCP-1 mRNA levels (Fig. 3A). The addition of TLR5 ligand
(flagellin) further increased MCP-1 and KC mRNA levels un-
der these conditions, and this occurred in a flagellin dose-
dependent manner (Fig. 3B and C). On the other hand, che-
mokine gene expression responses to TLR9 ligand (CpG
DNA) were not enhanced by prior exposure to IFN-?, al-
though stimulation of murine monocytes with the same CpG
DNA resulted in robust upregulation of MCP-1 and KC, thus
confirming its efficacy (data not shown). Overall, these findings
indicate that TLR2 and TLR4 ligands, as well as TLR5 ligand
in the setting of IFN-? priming, are able to signal both CC and
CXC chemokine gene expression in differentiated skeletal
Role of NF-?B in TLR-mediated chemokine expression by
skeletal muscle cells. Because TLR2 and TLR4 stimulation
produced the most prominent effects on MCP-1 and KC ex-
pression, subsequent experiments were focused upon these two
receptors. To compare the relative abilities of TLR2 and TLR4
to induce NF-?B transcriptional activity in cultured myotubes,
an expression plasmid containing an NF-?B luciferase reporter
was transfected into muscle cells prior to TLR stimulation.
Figure 4A indicates that NF-?B reporter values were signifi-
cantly increased above basal values after engagement of either
FIG. 1. Expression of TLRs by skeletal muscle. RT-PCR of RNA
extracted from the C2C12 muscle cell line at the myoblast (Mb) and
myotube (Mt) stages, as well as C57BL/10 mouse diaphragm, limb
muscle (tibialis anterior), and corresponding primary muscle cell cul-
tures (myotube stage). PCRs were also performed without addition of
reverse transcriptase (?CL) and in murine monocytes (?CL).
VOL. 74, 2006 TLRs AND CHEMOKINES IN SKELETAL MUSCLE6831
TLR2 or TLR4 with their respective ligands. However, TLR4
stimulation resulted in luciferase activity that was an order of
magnitude larger than that achieved by TLR2 stimulation. It
should be noted that the ligand concentrations employed (10
?g/ml PGN for TLR2; 1 ?g/ml LPS for TLR4) represented
maximal doses with respect to chemokine mRNA induction.
This is demonstrated in Fig. 4B, which demonstrates equiva-
lent MCP-1 and KC mRNA expression levels in response to
either ligand at a 10-fold lower dose.
We next sought to determine whether inhibition of NF-?B
signaling would attenuate chemokine gene expression induced
by either TLR2 or TLR4 stimulation. Therefore, the effects of
a pharmacological inhibitor of NF-?B (PDTC) on MCP-1 and
KC mRNA levels were examined, as shown in Fig. 5A. Treat-
ment of the cells with PDTC significantly reduced chemokine
mRNA levels in C2C12 myotubes exposed to PGN or LPS.
This did not appear to be mediated through effects on free
radical species, since treatment of the cells with two different
antioxidants, N-acetylcysteine (NAC) and catalase, did not
have any significant effects upon chemokine gene expression
under the same conditions. Interestingly, the effects of PDTC
treatment on KC mRNA levels were more pronounced for
TLR4 than TLR2 stimulation (Fig. 5B and C). In addition,
while PDTC treatment in the setting of TLR4 stimulation led
to a dramatic decrease in MCP-1 mRNA, the MCP-1 expres-
sion level during TLR2 stimulation was not significantly
affected by PDTC treatment.
To further evaluate the role of NF-?B in TLR2- and TLR4-
mediated chemokine gene expression, muscle cells were stim-
ulated with TLR ligands in the presence or absence of a dom-
inant-negative form of IKK?. In response to TLR activation,
IKK? normally phosphorylates cytoplasmic I?B, thereby tar-
geting the protein for ubiquitination and degradation, which
then permits the release and nuclear translocation of NF-?B.
Immunoblotting was used to monitor changes in total and
phospho-I?B? protein levels after TLR2 and TLR4 stimula-
tion as well as to determine the potential of the dominant-
negative IKK? construct to interfere with this process. As
shown in Fig. 6, both PGN and LPS stimulation induced rapid
I?B? phosphorylation in muscle cells transfected with a con-
trol plasmid. This was associated with a decrease in total I?B?,
which persisted for at least 60 min after stimulation with either
PGN or LPS. Notably, these responses were more rapid as well
as increased in magnitude after LPS compared to PGN expo-
sure, again suggesting stronger activation of the NF-?B path-
way with TLR4 than with TLR2 stimulation.
Transfection with the dominant-negative form of IKK? was
able to abrogate I?B phosphorylation/degradation to a very
large extent in both PGN- and LPS-stimulated muscle cells
(Fig. 6). Furthermore, Fig. 7 shows that transfection with the
dominant-negative IKK? construct significantly reduced both
MCP-1 and KC mRNA in myotubes exposed to LPS. However,
during PGN exposure, dominant-negative inhibition of IKK?
had lesser effects on KC expression, and MCP-1 expression was
entirely unaffected, in a manner that closely resembled the
results obtained during PGN stimulation in the presence of
PDTC. Overall, these observations are consistent with a
greater role for IKK? and the NF-?B signaling pathway in
TLR4- than in TLR2-mediated MCP-1 and KC gene expres-
sion by skeletal muscle cells.
TLR-mediated chemokine expression by skeletal muscle
cells involves calcineurin. We next employed FK506 to probe
the role of calcineurin in the observed MCP-1 and KC re-
sponses to TLR2 and TLR4 stimulation. Treatment of myo-
tubes with FK506, a calcineurin inhibitor, resulted in a small
but statistically significant reduction in LPS-induced KC mRNA
FIG. 2. TLR-mediated chemokine expression by skeletal muscle cells. RNase protection assays performed 4 h after incubation of C2C12
myotubes with S. aureus peptidoglycan (10 ?g/ml), poly(I:C) (25 ?g/ml), E. coli LPS (1 ?g/ml), S. enterica serovar Typhimurium flagellin (1 ?g/ml),
loxoribine (1 mM), or unmethylated CpG motif oligonucleotide (1 ?M), representing TLR 2, 3, 4, 5, 7, and 9 ligands, respectively. ?CL,
unstimulated C2C12 myotubes.
6832BOYD ET AL.INFECT. IMMUN.
levels, whereas no significant effects on LPS-induced MCP-1
expression were observed (Fig. 8A and B). In contrast, FK506
greatly reduced the magnitude of both MCP-1 and KC mRNA
levels induced by TLR2 stimulation with PGN (Fig. 8A and C).
Therefore, these data suggest a relatively greater role for cal-
cineurin signaling in the process of TLR2-mediated chemokine
gene expression by skeletal muscle. In addition, to verify the
specificity of TLR2 in mediating PGN effects on skeletal mus-
cle cell chemokine expression, primary myotubes derived from
TLR2 null mutant mice were also stimulated with either PGN
or LPS. As shown in Fig. 8D, PGN-mediated chemokine re-
sponses were abolished in myotubes lacking TLR2, whereas
responsiveness to LPS was preserved.
Skeletal muscle weakness occurs during severe infections
and may even result in the development of respiratory failure
and death due to weakness of the diaphragm and other respi-
ratory muscles (5, 14, 24, 29). The CC chemokines such as
MCP-1 are classically considered to be chemotactic factors for
mononuclear cells, whereas KC belongs to the ELR motif
(glutamate-leucine-arginine)-positive group of CXC chemo-
kines involved in neutrophil recruitment (34). We have re-
cently found that both CC and CXC chemokines are highly
upregulated in the septic mouse diaphragm in vivo (12), and
infiltration of the diaphragm by macrophages and neutrophils
during sepsis has been previously implicated in the pathogen-
esis of sepsis-induced diaphragmatic weakness (5, 29, 42). In-
creased chemokine expression, leukocyte infiltration, and
weakness are also found in the diaphragm and other muscles of
mice suffering from muscular dystrophy (11). Therefore, it is
reasonable to speculate that chemokine expression by skeletal
muscles is likely to play a significant role in the pathogenesis of
In the present investigation, we observed that differentiated
myotubes (from C2C12 cells or primary cells) and intact whole
muscles all expressed TLR2, TLR4, TLR5, and TLR9. Our
TLR expression pattern findings are generally consistent with
those of other investigators, although there are some differ-
ences. Frost and colleagues have reported that TLRs 1 to 7
(but not TLR8 or TLR9) are expressed in C2C12 myotubes
(16, 17). This differs from our own findings with respect to
TLRs 3 and 7 (not detected in our study) as well as TLR9
(detected in our study). However, these authors also reported
a failure to achieve functional responses (IL-6 expression)
upon stimulation with TLR3 and TLR9 ligands (17), which is
in agreement with the results of our study. Two other groups
have recently reported TLR9 mRNA expression in human
skeletal muscle tissue (32) and human myoblasts (38). The
latter study also found TLR3 expression in cultured human
myoblasts and in diseased muscles from myopathic patients but
FIG. 3. IFN-? priming of skeletal muscle cells augments chemokine expression in response to TLR5 stimulation. (A) RNase protection assay
demonstrating chemokine expression by C2C12 myotubes under the following conditions: unstimulated cells (CL), cells primed for 24 h with IFN-?
(IFN; 200 U/ml), cells stimulated for 4 h with flagellin (Fl) alone, and IFN-primed cells stimulated for 4 h with flagellin (IFN ? Fl). (B and C)
Quantification of chemokine mRNA levels by densitometry, expressed in arbitrary units (a.u.) and normalized to the L32 housekeeping gene (n ?
4 per group).*, P ? 0.05 versus IFN alone; †, P ? 0.05 for comparisons between IFN ? Fl 0.01 ?g/ml and IFN ? Fl 1.0 ?g/ml.
VOL. 74, 2006 TLRs AND CHEMOKINES IN SKELETAL MUSCLE 6833
not in normal healthy muscle fibers (38). It is likely that the
slightly divergent findings among these investigations is related
at least in part to differences in tissue culture conditions (e.g.,
duration and stage of differentiation, etc.) as well as possible
To our knowledge, the present study is the first to system-
atically evaluate the ability of the different TLRs to mediate
CC and CXC chemokine expression by skeletal muscle cells.
We assessed a broad range of different TLR-ligand interac-
tions and noted a complex pattern in which certain TLRs
expressed by the cells responded to stimulation in a straight-
forward manner (TLRs 2 and 4), while others required im-
mune modulation by another molecule to respond (IFN-? for
TLR5) or did not respond at all (TLR9). The ability of IFN-?
to potentiate TLR responses (as shown here for TLR5) is well
described in other cell types (19, 47). Therefore, one possible
reason for the lack of responsiveness to TLR9 ligand in our
study is a similar dependence upon another as yet unidentified
immunostimulatory molecule. In addition, while many cell
types respond when CpG DNA is applied at the cell surface
(22), the subcellular location of TLR9 is largely endosomal (26,
28). It is thus possible that skeletal muscle cells require addi-
tional measures or a more prolonged period of exposure to
achieve intracellular uptake of CpG DNA and effective TLR9
Not surprisingly, we observed that the NF-?B pathway is
activated after both TLR2 and TLR4 stimulation of skeletal
muscle cells. However, a novel finding in our study was the
documentation of major differences in the degree of NF-?B
activation induced by TLR2 versus TLR4 stimulation of skel-
etal muscle cells. Hence, at TLR ligand doses which were
supramaximal with respect to chemokine gene induction,
FIG. 4. TLR2 and TLR4 stimulation induce different levels of
NF-?B luciferase reporter activity. (A) C2C12 cells were cotransfected
with a NF-?B firefly luciferase reporter plasmid and a constitutively
active Renilla luciferase plasmid to control for transfection efficiency.
Myotubes were stimulated for 4 h with LPS (1 ?g/ml) or PGN (10
?g/ml) (n ? 4 per group).*, P ? 0.05 versus control (CL); †, P ? 0.05
for comparisons between PGN and LPS. (B) RNase protection assay
demonstrating that equivalent levels of KC and MCP-1 mRNA expres-
sion were observed in response to both ligands over a 10-fold dose
FIG. 5. Chemical inhibition of NF-?B signaling decreases TLR-
mediated chemokine expression in a differential manner. (A) RNase
protection assay demonstrating chemokine expression by C2C12 myo-
tubes after LPS or PGN stimulation for 4 h in the presence or absence
of the NF-?B inhibitor PDTC (100 ?M) and the antioxidant NAC (10
mM) or catalase (2,000 units/ml). (B and C) Quantification of chemo-
kine mRNA levels by densitometry, expressed in arbitrary units (a.u.)
and normalized to the L32 housekeeping gene (n ? 4 per group).*,
P ? 0.05 versus LPS or PGN alone.
6834 BOYD ET AL.INFECT. IMMUN.
TLR4 stimulation induced more rapid and complete I?B deg-
radation than was observed after TLR2 stimulation. In keeping
with these findings, TLR4 stimulation was also associated with
dramatically higher NF-?B luciferase reporter activity. In ad-
dition, interference with NF-?B activation during TLR4 stim-
ulation had greater inhibitory effects upon chemokine gene
expression. The compound PDTC, which blocks the E3 ubiq-
uitin ligase involved in I?B? degradation (20), greatly reduced
the levels of both KC and MCP-1 expression triggered by
TLR4 stimulation but had no effect upon MCP-1 expression
induced by TLR2 stimulation. Because PDTC also has antiox-
idant properties, and chemokine expression can be increased
by mechanisms which are dependent upon reactive oxygen
species (27), we also evaluated the effects of free radical scav-
engers. We employed two potent antioxidants, NAC and cata-
lase, with documented abilities to inhibit reactive oxygen spe-
cies-mediated cytokine upregulation in muscle cells at the
doses employed in this study (25). In contrast to PDTC, these
antioxidants had no significant effects upon TLR2- or TLR4-
mediated chemokine gene expression, suggesting that free rad-
icals were not responsible for chemokine induction in our
experiments. Furthermore, as in the case of PDTC, dominant-
negative IKK? significantly inhibited KC and MCP-1 expres-
sion induced by TLR4 stimulation, whereas for TLR2 stimu-
lation, it had substantially smaller effects upon KC and no
impact at all upon MCP-1 expression. Therefore, it appears
that NF-?B activation is greater in magnitude during TLR4
stimulation and also plays a more important role in TLR4-
than in TLR2-mediated chemokine expression by skeletal mus-
It is important to note that, despite a significantly lower level
of NF-?B activation in the case of TLR2 stimulation, the mag-
nitude of MCP-1 and KC mRNA expression was equally robust
when cells were exposed to supramaximal doses of either PGN
or LPS. This finding indicates that TLR2 and TLR4 must
recruit different signaling pathways to regulate the same che-
mokine genes in skeletal muscle. A plausible candidate for
another signaling molecule is the calcium-dependent serine/
threonine phosphatase calcineurin, which is known to be ex-
pressed by skeletal muscle cells, where its actions in favoring
the nuclear translocation of NFAT have been implicated in the
regulation of skeletal muscle growth, differentiation, and spe-
cialization (9, 31, 40). In activated T lymphocytes, calcineurin
inhibition by FK506 has been found to have differential effects
on chemokines, with several CC chemokines being repressed
to variable degrees (MIP-1?, MIP-1?, RANTES), whereas a
CXC chemokine (IP-10) actually demonstrated upregulation
(41). In addition, calcineurin has been reported to upregulate
MCP-1 expression in vascular smooth muscle cells by augment-
ing mRNA stability (36).
Here we show for the first time that calcineurin plays a major
role in the regulation of chemokine expression by skeletal
muscle cells. In this regard, we found that TLR2-mediated
induction of both MCP-1 and KC was markedly attenuated by
the calcineurin inhibitor FK506. On the other hand, during
TLR4 stimulation, FK506 had a less pronounced inhibitory
influence on KC expression, and no significant effect on
MCP-1 expression could be demonstrated. Therefore, the im-
pact of calcineurin inhibition on chemokine expression was
substantially greater for TLR2- than TLR4-mediated re-
sponses, which is in direct contradistinction to the results ob-
tained during NF-?B inhibition as discussed earlier. For
MCP-1 in particular, there was a high degree of TLR-depen-
dent differential regulation by NF-?B and calcineurin. Our
findings are reminiscent of recent observations in human air-
way epithelial cells, in which it was reported that another
FIG. 6. TLR2 and TLR4 ligands induce I?B? phosphorylation and degradation in skeletal muscle cells. Immunoblot analysis of phosphorylated
I?B? (P-I?B) and total I?B? (I?B) in C2C12 myotubes transfected with a dominant-negative IKK? plasmid vector or an empty control vector
containing the same plasmid backbone and stimulated with LPS (1 ?g/ml) or PGN (10 ?g/ml).
VOL. 74, 2006 TLRs AND CHEMOKINES IN SKELETAL MUSCLE6835
calcineurin inhibitor, cyclosporine, achieved almost complete
abrogation of TLR2-mediated induction of IL-8 (CXCL8) ex-
pression, whereas responses to heat-killed gram-negative bac-
teria (presumably TLR4 mediated to a large extent) could be
inhibited by about 25% only (51). This suggests that cal-
cineurin may play a predominant role in the mediation of
TLR2-stimulated chemokine responses not only within skeletal
muscle but within other cell types as well.
It is of interest to consider the potential effects of calcineurin
on several different transcription factors which could be in-
volved in MCP-1 or KC gene regulation. One possibility would
be an effect of calcineurin on NF-?B itself (46), particularly
since forced overexpression of activated calcineurin in C2C12
cells has been reported to increase NF-?B activity (2), and
mitochondrial stress can also lead to NF-?B activation in
C2C12 cells through a calcineurin-dependent mechanism (4).
However, FK506 produced substantially less inhibition of che-
mokine expression during stimulation of TLR4 relative to
TLR2, despite the fact that NF-?B activity during TLR4 stim-
ulation was an order of magnitude higher than for TLR2.
Therefore, it seems unlikely that the major effects of cal-
cineurin inhibition observed during TLR2 stimulation were
mediated through an effect on NF-?B. Calcineurin also has the
potential to modulate several other proinflammatory signaling
pathways which could be involved in TLR-mediated chemo-
FIG. 7. Dominant-negative molecular inhibition of NF-?B signal-
ing decreases TLR-mediated chemokine expression in a differential
manner. (A) RNase protection assay demonstrating chemokine
mRNA responses to LPS or PGN stimulation after transfection with
either dominant-negative IKK? (IKKB DN) or control plasmid vector
as described in the legend to Fig. 6. (B and C) Quantification of
chemokine mRNA levels by densitometry, expressed in arbitrary units
(a.u.) and normalized to the L32 housekeeping gene (n ? 4 per group).
*, P ? 0.05 versus control vector.
FIG. 8. The calcineurin inhibitor FK506 differentially affects
MCP-1 and KC gene expression in a TLR-specific manner. (A) RNase
protection assay demonstrating the effects of FK506 (100 ng/ml) on
chemokine expression by C2C12 myotubes after LPS or PGN stimu-
lation for 4 h (together with the vehicle used for FK506, 0.1 ?l etha-
nol/ml of media). (B and C) Corresponding quantification of chemo-
kine mRNA levels by densitometry;*, P ? 0.05 versus LPS or PGN
alone. (D) Quantification of increases in chemokine mRNA levels in
primary myotubes derived from wild-type (WT, C57BL/6) and TLR2
null mutant mice following stimulation with either LPS or PGN for 4 h
(n ? 4 per group).*, P ? 0.05 versus unstimulated control.
6836 BOYD ET AL.INFECT. IMMUN.
kine gene expression by skeletal muscle cells, such as NFAT,
CREB, and C/EBP (18, 31, 39). The latter, in particular, has
been strongly implicated in transcriptional regulation of both
MCP-1 (1, 39) and KC (7) gene expression.
Recently, it has become apparent that chemokines have
important biological functions in skeletal muscle which extend
well beyond their classical roles as leukocyte chemoattractants.
In particular, there is accumulating evidence for a significant
role in muscle regeneration following injury (49, 50). In animal
models of acute and subacute sepsis, skeletal muscle fiber
injury has been demonstrated (14, 29). Recovery from injury
involves myoblast precursors (called satellite cells) that are
normally quiescent in adult skeletal muscle but which become
activated to proliferate and migrate to form new muscle fibers
(regeneration) when skeletal muscle is damaged (23). It has
recently been demonstrated that CCR2 (the major receptor for
MCP-1) is expressed by skeletal muscle cells in vivo, and both
CCR2 and MCP-1 are required for optimal functional recovery
from injury (49, 50). In addition, RANTES (CCL5) has been
shown to be a chemotactic factor for myoblasts (8), and the
CXC chemokine LIX (LPS-induced CXC chemokine) is ex-
pressed in satellite cells shortly after induced muscle injury
(35). Therefore, although the blocking of chemokine expres-
sion in muscle during sepsis could mitigate leukocyte-mediated
adverse effects, there is also the potential for interference with
muscle repair mechanisms. Accordingly, the rational design of
therapeutic interventions in this area will require a detailed
understanding of the roles played by specific chemokines in
skeletal muscle during different stages of sepsis and recovery
from injury. Finally, in view of the possible role of TLRs in
sensing other types of cellular injury or stress beyond those
associated with infectious insults (48), it will be of considerable
interest in future studies to determine whether TLR-mediated
signaling plays a role in the augmented skeletal muscle che-
mokine gene expression found in noninfectious pathological
conditions such as the muscular dystrophies and inflammatory
This study was supported by the Canadian Institutes of Health
Research, the Respiratory Health Network of the Fonds de la Recher-
che en Sante du Quebec, and the Burroughs Wellcome Fund.
We thank J. Marshall and S. Akira for providing the use of TLR2
null mutant mice.
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Editor: J. F. Urban, Jr.
6838BOYD ET AL.INFECT. IMMUN.