Both TLR2 and TRIF Contribute to Interferon-b
Production during Listeria Infection
Camille Aubry1,2,3,4.¤a, Sine ´ad C. Corr5., Sebastian Wienerroither6., Ce ´line Goulard1,2,3¤b, Ruth Jones5,
Amanda M. Jamieson6, Thomas Decker6, Luke A. J. O’Neill5, Olivier Dussurget1,2,3,4*, Pascale Cossart1,2,3*
1Institut Pasteur, Unite ´ des Interactions Bacte ´ries-Cellules, Paris, France, 2Inserm, U604, Paris, France, 3INRA, USC2020, Paris, France, 4Universite ´ Paris Diderot, Sorbonne
Paris Cite ´, Cellule Pasteur, Paris, France, 5School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Ireland, 6Max F. Perutz
Laboratories, Department of Genetics, Microbiology and Immunobiology, University of Vienna, Vienna, Austria
Synthesis of interferon-b (IFN-b) is an innate response to cytoplasmic infection with bacterial pathogens. Our recent studies
showed that Listeria monocytogenes limits immune detection and IFN-b synthesis via deacetylation of its peptidoglycan,
which renders the bacterium resistant to lysozyme degradation. Here, we examined signaling requirements for the massive
IFN-b production resulting from the infection of murine macrophages with a mutant strain of L. monocytogenes, DpgdA,
which is unable to modify its peptidoglycan. We report the identification of unconventional signaling pathways to the IFN-b
gene, requiring TLR2 and bacterial internalization. Induction of IFN-b was independent of the Mal/TIRAP adaptor protein but
required TRIF and the transcription factors IRF3 and IRF7. These pathways were stimulated to a lesser degree by wild-type L.
monocytogenes. They operated in both resident and inflammatory macrophages derived from the peritoneal cavity, but not
in bone marrow-derived macrophages. The novelty of our findings thus lies in the first description of TLR2 and TRIF as two
critical components leading to the induction of the IFN-b gene and in uncovering that individual macrophage populations
adopt different strategies to link pathogen recognition signals to IFN-b gene expression.
Citation: Aubry C, Corr SC, Wienerroither S, Goulard C, Jones R, et al. (2012) Both TLR2 and TRIF Contribute to Interferon-b Production during Listeria
Infection. PLoS ONE 7(3): e33299. doi:10.1371/journal.pone.0033299
Editor: Dario S. Zamboni, University of Sa ˜o Paulo, Brazil
Received September 16, 2011; Accepted February 7, 2012; Published March 14, 2012
Copyright: ? 2012 Aubry 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: Work in PC’s laboratory received financial support from Institut Pasteur (http://www.pasteur.fr), Inserm (http://www.inserm.fr), INRA (http://www.inra.
fr), ERC (Advanced Grant 233348, http://erc.europa.eu/), Fondation Pasteur-Weizmann and Fondation le Roch Les Mousquetaires (http://www.fondationleroch-
lesmousquetaires.org/). PC is an international research scholar of the Howard Hughes Medical Institute (http://www.hhmi.org/). CA is a doctoral fellow of the
Ministe `re de l’Enseignement Supe ´rieur et de la Recherche. Work in LO’s laboratory was supported by Science Foundation Ireland (http://www.sfi.ie/) and the Irish
Research Council for Science, Engineering and Technology (RS/2005/190, http://www.ircset.ie/). Work in TD’s laboratory was funded by the Austrian Research
Foundation (grants SFB-28 and P20522-B05, http://www.fwf.ac.at). 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: email@example.com (PC); firstname.lastname@example.org (OD)
. These authors contributed equally to this work.
¤a Current address: Interactions des Bacte ´ries Commensales et Probiotiques avec l’Ho ˆte, INRA-MICALIS, Jouy-en Josas, France
¤b Current address: EA3647, Universite ´ de Versailles St-Quentin-en-Yvelines et Laboratoire de Microbiologie, Ho ˆpital Raymond Poincare ´, Assistance Publique -
Ho ˆpitaux de Paris, Garches, France
Detection of microbial pathogens by pattern recognition
receptors, such as Toll-like receptors (TLRs) triggers innate
immune responses as a first line of defense against infections [1–
3]. Pathogen-associated molecular patterns (PAMPs) such as
bacterial cell walls and their structural components induce a vast
variety of biological effects in host organisms. The innate response
against infection with intracellular pathogens includes the synthesis
of type I IFNs (IFN-I). Whereas this cytokine family generally
protects against viruses, its impact on bacterial infections can be
either detrimental or advantageous for the host organism .
Listeria monocytogenes is a bacterial pathogen which replicates in the
cytoplasm of infected cells. Cytosolic pattern recognition receptors
(PRRs) respond to cytosolic bacterial products and contribute to the
induction of the innate immune response [5,6]. Previous studies in
bone marrow-derived macrophages (BMM) and epithelial cells
show that in these cell types the synthesis of IFN-I in response to
infection with L. monocytogenes is independent of TLRs and their
adapters, relying exclusively on signals originating from cytosolic
sensors [5–8]. DNA as well as cyclic dinucleotides released from
lysed bacteria were suggested to function as the relevant L.
monocytogenes PAMPs [9–11]. Several cytosolic proteins with the
ability to sense pathogen-derived nucleic acids have recently been
described [11–19]. Cytosolic recognition of L. monocytogenes causes
the activation of the serine/threonine kinase TBK1 and the
phosphorylation of its substrate transcription factors IRF3 and
IRF7 [7,8]. Both IRF3 and IRF7 participate in the formation of an
enhanceosome at the IFN-b promoter .
During uptake by host cells L. monocytogenes is exposed to plasma
membrane and endosomal TLRs. Among these, TLR2 which
recognizes lipotechoic acids and lipopeptides, contributes to the
innate response against infection [21–23]. Reportedly, TLR2
signals through the interacting adapter proteins Mal/TIRAP and
MyD88 and does not contribute to the synthesis of type I IFN in
Listeria-infected BMM [7–9]. Signaling through TRIF, an adapter
protein known to connect TLRs 3 and 4 with the IFN-I genes was
similarly ruled out for Listeria-infected BMM .
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In order to establish a successful infection, pathogens must
survive host defense systems or else mitigate the activities of PRRs.
Consequently, they have evolved to modify the structural
components which normally trigger PRR responses. Bacterial
PGN is a hetero-polymer consisting of alternating residues of b-
1,4-linked N-acetylglucosamine and N-acetylmuramic acid to
which a peptide chain is attached . Interestingly, L.
monocytogenes modifies its PGN, with fifty per cent of the
muropeptide composition being N-deacetylated . We previ-
ously reported that a PGN N-deacetylase gene, pgdA, is responsible
for this modification . PGN deacetylation confers resistance to
the action of lysozyme, one of the most important and widespread
antimicrobial agents of the innate defense system, thus preventing
degradation and release of immunostimulants. A strain of L.
monocytogenes mutated in its ability to alter its PGN, DpgdA, is
sensitive to lysozyme and induces an enhanced IFN-b response in
macrophages compared to the isogenic parental strain .
The aim of the present study was to decipher the signaling
pathways involved in this response to DpgdA infection. We reveal
that IFN-b production in peritoneal macrophages requires TLR2
signaling and the TRIF adapter protein.
IFN-b is highly expressed in response to infection with
Listeria DpgdA mutant in a TLR2-dependent manner
A L. monocytogenes pgdA mutant induced a much higher IFN-b
response than the parental strain . To definitively establish a
role for the peptidoglycan deacetylase PgdA in the down-
regulation of IFN-b production, we complemented our original
pgdA mutant with the wild-type gene and we measured IFN-b
secretion of peptone elicited peritoneal macrophages (PEM)
infected with wild-type EGDe, DpgdA and a complemented DpgdA
strain (Fig. 1). Inactivation of pgdA led to a strong induction of IFN-
b secretion in wild-type macrophages. In contrast, the comple-
mented strain did not induce any massive IFN-b secretion, similar
to wild-type EGDe. Thus, PgdA directly contributes to down-
regulation of IFN-b production.
Consistent with our previous report measuring secretion of IFN-
b protein in PEM, IFN-b mRNA synthesis induced by L.
monocytogenes infection of PEM required TLR2 (Fig. 2A), while
TLR2-deficient BMM showed no impairment in their synthesis of
IFN-b mRNA (Fig. 2B). Moreover, IFN-b secretion was strongly
reduced in tlr22/2PEM infected with both the DpgdA mutant
(Fig. 2C) and the complemented DpgdA strain (Fig. 2D), definitively
establishing the TLR2 dependence of IFN-b production.
IFN-b induction does not require Mal/TIRAP but depends
We next analyzed the pathways by which Listeria induces IFN-b.
Our previous study and the above results strongly suggested the
critical involvement of TLR2 . TLR2 signaling depends on
Mal/TIRAP and MyD88 adaptor proteins. We had previously
shown that MyD88 contributed to full IFN-b induction by Listeria
. We then compared IFN-b production by wild-type and mal/
tirap2/2macrophages infected with EGDe or DpgdA (Fig. 3A).
Surprisingly, production of IFN-b was not decreased in infected
macrophages deficient in Mal/TIRAP, indicating that the normal
TLR2 adaptor Mal/TIRAP was not required for Listeria-mediated
induction of IFN-b.
The adapter TRIF is employed by TLRs 3 and 4 to signal
through the TBK1-IRF3/7-IFN-b pathway. There is no previous
evidence of an association or functional interaction between TRIF
Figure 1. PgdA-dependent IFN-b response to Listeria in
peritoneal macrophages. PEM from WT C57BL/6J mice were
infected with the parental EGDe strain (black bars), the DpgdA mutant
(grey bars) or the complemented DpgdA strain (white bars). After 7 h of
infection, IFN-b levels were measured in supernatants by ELISA. Data are
mean 6 SD (***, p,0.0001, n=5).
Figure 2. TLR2 is required for PgdA-mediated IFN-b response to Listeria in peritoneal but not bone-marrow macrophages. (A) PEM
from C57BL/6J or tlr22/2mice were infected with the parental EGDe strain. After 4 h of infection, IFN-b induction was measured by qRT-PCR. (B) BMM
from C57BL/6J or tlr22/2mice were infected with the parental EGDe strain. After 4 h of infection, IFN-b induction was measured by qRT-PCR. Data are
mean 6 SD (NS, non significant; ***, p,0.0001, n=3). PEM from WT C57BL/6J or tlr22/2mice were infected with the DpgdA mutant (C) or the
complemented DpgdA strain (D). After 7 h of infection, IFN-b levels were measured in supernatants by ELISA. Data are mean 6 SD (**, p,0.01, n=5).
Listeria Induces IFN-b through TLR2 and TRIF
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and TLR2. In spite of this, the link between TRIF and the IRF
pathway on the one hand, and the unusual employment of TLR2
for signaling to the IFN-b gene in PEM on the other suggested the
possibility of a role for TRIF. To test this hypothesis we compared
induction of IFN-b expression in wild-type and trif2/2PEM or
BMM infected with EGDe or DpgdA strains. IFN-b induction
strongly decreased in TRIF-deficient macrophages infected with
any of the two Listeria strains compared to wild-type PEM, showing
the requirement for TRIF (Fig. 3B). In contrast, BMM showed a
TRIF-independent IFN-b production (Fig. S1).
The PEM used in our studies are recruited to the peritoneal
cavity by injection of the sterile irritant proteose peptone. Hence
they differ from BMM not only regarding their anatomical location,
but also their partially inflammatory character. To distinguish
which of these differences was responsible for the TLR2 and TRIF
signaling pathways, we examined IFN-b production by resident
PEM. Figure 3C demonstrates a requirement for TLR2 and TRIF
by the resident macrophage population. Thus, location to the
peritoneal cavity rather than inflammatory character determines
the difference in signaling to the IFN-b gene between BMM and
To examine the role of TLR3, which uses TRIF to trigger IFN-
b synthesis, we compared induction of IFN-b in wild-type and
tlr32/2PEM infected with EGDe or DpgdA strains. IFN-b
production was decreased in TLR3-deficient PEM infected with
EGDe or DpgdA (Fig. 4A). We also compared induction of IFN-b
in wild-type and tlr42/2PEM infected with EGDe or DpgdA
strains, as TLR4 can mediate TRIF-dependent synthesis of IFN-b.
In contrast to TLR3-deficient PEM, TLR4-deficient PEM did not
show a decrease in IFN-b response to EGDe or DpgdA (Fig. 4B).
Thus, IFN-b induction in response to Listeria infection relies in part
on TLR3 and does not require TLR4.
IFN-b is induced by intracellular bacteria
Induction of IFN-b via TLR2 is no longer an exception. It has
recently been shown that vaccinia virus-induced IFN-b production
Figure 3. TRIF, but not Mal/TIRAP, is necessary for IFN-b response to Listeria in peritoneal macrophages. (A) PEM from WT C57BL/6J or
mal/tirap2/2mice were infected with the parental EGDe strain (black bars), the DpgdA mutant (grey bars). After 7 h of infection, IFN-b levels were
measured in supernatants by ELISA. (B) PEM from C57BL/6J or trif2/2mice were infected with the parental EGDe strain (black bars) or the DpgdA
mutant (grey bars). After 4 h of infection, IFN-b induction was measured by qRT-PCR. (C) Resident peritoneal macrophages from WT C57BL/6J, trif2/2
or tlr22/2mice were infected with the parental EGDe strain (black bars), the DpgdA mutant (grey bars). After 4 h of infection, IFN-b induction was
measured by qRT-PCR. Data are mean 6 SD (NS, non significant; *, p,0.05; ***, p,0.0001; n=3–4).
Figure 4. TLR3, but not TLR4, contributes to IFN-b response to Listeria in peritoneal macrophages. (A) PEM from WT C57BL/6J or tlr32/2
mice were infected with the parental EGDe strain (black bars), the DpgdA mutant (grey bars). After 7 h of infection, IFN-b levels were measured in
supernatants by ELISA. Data are mean 6 SD (n=3). (B) PEM from WT C57BL/6J or tlr42/2mice were infected with the parental EGDe strain (black
bars), or the DpgdA mutant (grey bars). After 7 h of infection, IFN-b levels were measured in supernatants by ELISA. Data are mean 6 SD
(***, p,0.0001; n=3).
Listeria Induces IFN-b through TLR2 and TRIF
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was dependent on TLR2 signaling and it was reported that this
was occuring from late endosomes . To investigate if an
intracellular localization was also required in the case of Listeria, we
pretreated cells with cytochalasin D to prevent internalization and
measured IFN-b secretion by macrophages infected with EGDe or
the DpgdA mutant (Fig. 5A). In both cases, IFN-b induction was
strongly reduced. Thus, internalization is critical for Listeria-
mediated IFN-b production. We also used dynasore, a dynamin
inhibitor and chloroquine, which inhibits endosome acidification,
and measured IFN-b induction in macrophages infected with
EGDe or the DpgdA mutant (Fig. 5B–C). IFN-b synthesis was
strongly diminished by both dynasore and chloroquine treatments.
Together, these results suggest that the TLR2-dependent IFN-b
induction is triggered intracellularly.
IRF3 and IRF7 are essential for IFN-b production in
response to Listeria infection
In BMM rapid synthesis of IFN-b is entirely dependent on IRF3,
but not on IRF7, whereas in bone marrow-derived myeloid DC
IFN-b synthesis requires both IRF3 and IRF7 . We investigated
the role of IRF3 and IRF7 in the production of IFN-b by PEM. To
this end we infected irf32/2and irf72/2macrophages with EGDe
or the DpgdA strains. Inactivation of IRF3 totally abrogated IFN-b
mRNA induction in response to both strains (Fig. 6). IFN-b
induction in IRF7-deficient macrophages was also strongly affected
highlighting the important role of both transcription factors in
response to Listeria infection (Fig. 6). PEM thus resemble bone
marrow-derived myeloid DC, not BMM, in relation to their IRF
requirement for Listeria-mediated IFN-b synthesis.
In addition to IRF3/7, NFkB contributes to the formation of
the IFN-b enhanceosome [20,28]. We therefore examined the
involvement of the NFkB pathway by measuring induced synthesis
of an NFkB-dependent mRNA. IkB is an NFkB-dependent gene
and thus a read-out for NFkB activation in response to Listeria
infection. We measured the induction of IkB expression in PEM
infected with EGDe or the DpgdA mutant. Both strains induced
IkB expression and this required internalization as treatment with
dynasore reduced the level of IkB induction (Fig. 7A). Degradation
of the IkB protein was examined in PEM infected with EGDe by
immunoblot using anti-IkB antibodies. IkB level was reduced
rapidly after infection of wild-type PEM (Fig. S2A). In contrast,
IkB degradation was not observed in tlr22/2PEM infected with
Listeria (Fig. S2B). Infection of wild-type, tlr22/2and trif2/2
Figure 5. Internalization of bacteria is required for IFN-b response by peritoneal macrophages. (A) PEM from WT C57BL/6J mice were
pretreated with 50 mM of cytochalasin D, and left uninfected (hatched bars) or infected with the parental EGDe strain (black bars) or the DpgdA
mutant (grey bars). 7 h post-infection, IFN-b levels were measured in cells supernatants by ELISA. (B) PEM from WT C57BL/6J were treated with 80 mM
dynasore. After 4 h of infection with the parental EGDe strain (black bars) or the DpgdA mutant (grey bars), IFN-b induction was measured by qRT-
PCR. (C) PEM from WT C57BL/6J mice were treated with 100 mM chloroquine, and left uninfected (hatched bars) or infected with the parental EGDe
strain (black bars) or the DpgdA mutant (grey bars). 7 h post-infection, IFN-b concentrations were measured in cells supernatants by ELISA. Data are
mean 6 SD (***, p,0.0001, n=3–4).
Figure 6. IFN-b response to Listeria is mediated by IRF3 and
IRF7 in peritoneal macrophages. PEM from WT C57BL/6J, irf32/2
and irf72/2mice were infected with the parental EGDe strain (black
bars) or the DpgdA mutant (grey bars). 4 h post-infection, mRNA was
isolated and the IFN-b induction was measured by qRT-PCR. Data are
mean 6 SD (***, p,0.0001, n=3).
Figure 7. Bacterial internalization and NF-kB are required for
TLR2 and TRIF-dependent IFN-b response in peritoneal mac-
rophages. (A) PEM from WT C57BL/6J mice and pretreated with
dynasore were infected with the parental EGDe strain (black bars) or the
DpgdA mutant (grey bars). 4 h post-infection, IkB induction was
measured by qRT-PCR. (B) PEM from WT C57BL/6J, tlr22/2and trif2/2
mice were infected with the parental EGDe strain (black bars) or the
DpgdA mutant (grey bars). 4 h post-infection, mRNA was isolated and
IkB induction was measured by qRT-PCR. Data are mean 6 SD
(***, p,0.0001, n=3).
Listeria Induces IFN-b through TLR2 and TRIF
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macrophages with EGDe or DpgdA showed that both TLR2 and
the adaptor were required for full induction of IkB mRNA in
response to EGDe and DpgdA strains (Fig. 7B). These results
suggest that TLR2 and TRIF contribute to NFkB activation. The
comparison between EGDe and DpgdA strains showed that both
caused similar magnitudes of IkB mRNA synthesis. Thus, the
activation of NFkB by Listeria is independent of PgdA, suggesting
that the increased IFN-b production after infection with DpgdA
relies on activation of other transcription factors such as IRFs.
Nucleic acids released intracellularly are critical for IFN-b
TLR2 or TRIF deficiency strongly reduced, but did not
completely shut off IFN-b synthesis. This suggested a potential
contribution of intracellular, nucleic acid-dependent pathways to
IFN-b synthesis, particularly after infection with DpgdA. We
therefore examined whether these pathways are able to signal in
Since inactivation of PgdA increases Listeria sensitivity to
peptidoglycan-targeting antimicrobials such as lysozyme, and thus
induces bacterial degradation, we measured the DNA and RNA
released by EGDe and DpgdA strains following lysozyme exposure.
As expected, DpgdA released significantly higher amounts of DNA
and RNA than wild-type and complemented DpgdA strains, raising
the possibility that both DNA and RNA could be involved in IFN-
b production (Fig. 8A). We thus measured IFN-b induction in
THP1 macrophages transfected with Listeria DNA, either
undigested or treated with DNase. Intact but not DNase-treated
DNA significantly induced IFN-b (Fig. 8B). Macrophages were
then transfected with lysozyme-digested EGDe or DpgdA, either
untreated or digested with DNase. Treatment with DNase
significantly reduced IFN-b production (Fig. 8C). Taken together,
these results show that Listeria DNA can induce IFN-b, strongly
indicating that destruction of DpgdA bacteria intracellularly
activates DNA sensors.
We had recently reported that a PGN modification involving a
N-deacetylase gene, pgdA, was playing a key role in L. monocytogenes
virulence . A DpgdA strain of L. monocytogenes which is unable to
modify its PGN, was shown to be extremely sensitive to the
bacteriolytic activity of lysozyme, normally found within macro-
phage vacuoles and its virulence was strongly attenuated .
Furthermore, this mutant induced a much higher TLR2-
dependent IFN-b response than the parental strain . We
hypothesised that this unconventional IFN-b response induced by
the pgdA mutant was due to an enhanced accessibility of bacterial
cell wall components to TLR2. Here we have shown that IFN-b
production requires bacterial internalization and is triggered by
Mal/TIRAP-independent pathways which involve TLR2, TRIF,
IRF3 and IRF7.
It was surprising to see a role for TLR2, as, based on results in
BMM and epithelial cells, type I IFNs production is usually not
known to result from TLR2 signaling [5–8]. Classical TLR2
signaling leads to NF-kB-dependent production of inflammatory
cytokines . However, in support of an unconventional role for
TLR2, recent studies reported roles for TLR2-dependent
induction of IFN-b in response to vaccinia virus or synthetic
ligands [26,29]. In the vaccinia virus study, a specific inflammatory
monocyte population -Ly6Chi- was shown to be the source of IFN-
b . In the present study we show that TLR2-dependent IFN-b
synthesis is a property of both resident and recruited inflammatory
PEM. Furthermore, the two previous studies documented that
TLR2 activation of type I IFN responses to TLR ligands occurs
within intracellular compartments, and that TLR2 signals from
the phagosome in response to viral infection or synthetic TLR2
ligands [26,29]. These results challenged the view that TLR2
signals solely from the plasma membrane. In our experiments, pre-
treatment of PEM with either cytochalasin D, an inhibitor of actin
polymerization and thus internalization, dynasore, an inhibitor of
the endocytic effector dynamin, or chloroquine, which inhibits
endosome acidification [30,31], significantly impaired the induc-
tion of IFN-b following Listeria infection, strongly suggesting that
phagocytosis of L. monocytogenes and intracellular location of TLR2
trigger this response. These observations also correlate with our
early hypothesis that the inflammatory response induced by DpgdA
is due to an enhanced release or accessibility of bacterial cell wall
components to TLR2.
Induction of the IFN-b gene was independent of the TLR
adapter Mal/TIRAP, but, unexpectedly required the TLR3/4
adapter TRIF. Francisella tularensis has recently been shown to
Figure 8. Listeria nucleic acids trigger IFN-b production. (A) The parental EGDe (black bars) DpgdA (grey bars) and complemented DpgdA strain
(hatched bars) were incubated with lysozyme. The amount of DNA and RNA released after treatment was quantified by spectrophotometry. (B) THP-1
macrophages were transfected with DNA from the DpgdA mutant, pretreated or not with DNase, and IFN-b induction was determined using the HEK-
blue assay. (C) The parental EGDe strain (black bars) or the DpgdA mutant (grey bars) were incubated with lysozyme. PEM were transfected with
bacterial lysates, pretreated with DNase or not treated, and IFN-b production was quantified in cells supernatants 24 h after transfection by ELISA.
Data are mean 6 SD (**, p,0.01, n=3; ***, p,0.0001, n=3).
Listeria Induces IFN-b through TLR2 and TRIF
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signal through TLR2 from the phagosome in a Mal/TIRAP
independent manner , and it was shown that Mal/TIRAP is
dispensable in TLR2 signaling at high concentrations of ligands
. Thus our study reinforces the view that TLR2 can act
independently from Mal/TIRAP. In addition our report suggests
a synergy between a TLR2 pathway and TRIF, an adapter
previously known to trigger the synthesis of pro-inflammatory
cytokines and type I IFNs upon engagement of TLR3 and TLR4.
TLR3 is known to bind viral dsRNA to induce secretion of type I
IFN and lead to control of viral infections [3,34,35]. To our
knowledge Chlamydia muridarum is the only bacterium reported to
induce a TLR3-dependent IFN-b response specifically in murine
oviduct epithelial cells . We tested whether the dual
requirement for TLR2 and TRIF resulted from a functional or
physical interaction between TLR2 and TLR3. In fact, IFN-b
production was reduced in TLR3-deficient macrophages, but
significantly less so than in trif2/2 PEM. Therefore, there is no
evidence for a putative TLR2/TLR3 interaction. Another
possibility to incorporate TRIF into the pathway stimulated in
PEM by Listeria would be a cooperation of TLR2 and TLR4. This
was ruled out by showing that Listeria-infected tlr42/2PEM
produced a similar amount of IFN-b as their wild-type
counterparts. TRIF could possibly orchestrate an additional
pathway. Along these lines, TRIF has recently been shown to be
required for IFN-b synthesis by dendritic cells upon activation of
the cytosolic receptor complex DDX1/DDX21/DDX36 by viral
Engagement of TLRs by various microbe-associated molecular
patterns induces activation and translocation to the nucleus of NF-
kB, IRF3, IRF7 and/or activator protein-1 (AP-1), which
collaborate to induce transcription of type I IFNs . We
addressed the role of these transcriptional activators in the IFN-b
response to wild-type Listeria and DpgdA, and revealed that
inactivation of IRF3 totally abrogated this response to both strains
while IFN-b induction was significantly but not totally impaired in
IRF7-deficient macrophages, indicating that both of these
transcription factors are required for induction of IFN-b following
infection with L. monocytogenes. We also assessed the involvement of
NF-kB in this response using induction of the IkB gene as a
readout. We observed an induction of IkB expression in
macrophages which was similar after infection with EGDe or
DpgdA. Thus, activation of NF-kB by Listeria is independent of
PgdA, strongly suggesting that the elevated IFN-b production by
the DpgdA mutant mostly relies on IRF3.
The increased IFN-b response to the DpgdA strain probably
results from the fact that within the phagosome, its lysozyme-
sensitive cell wall is degraded, releasing PAMPs able to interact
with TLR2 and other PRRs, including cytoplasmic ones. As recent
studies have highlighted novel DNA-sensing pathways in the
induction of type I IFNs [9,14–17,38], we thus also investigated
the involvement of bacterial nucleic acids in the IFN-b induction,
Firstly, we showed that inactivation of PgdA, which confers a
higher susceptibility to lysozyme, leads to increased release of
DNA. We then showed that DNA from L. monocytogenes can induce
IFN-b expression in PEM, suggesting that this macrophage
population employs cytoplasmic nucleic acid sensing similar to
macrophages or macrophage lines derived from different anatom-
ical locations [9,38]. Which -if any- of the recently described
nucleic acid sensors are used by PEM for the recognition of Listeria
DNA remains subject to future investigation. Nevertheless, other
bacterial components could participate in IFN-b production upon
infection with the DpgdA mutant. For example, the second
messenger molecule cyclic diadenosine monophosphate (c-di-
AMP), was shown to be secreted by Listeria multidrug efflux
pumps triggering type I IFN response  and could be involved
in the process.
In conclusion, this study describes a novel mechanism leading to
induction of type I IFNs in which intracellular sensing plays an
important role, ultimately showing how these different recognition
pathways can synergise to induce innate immune responses which
are required to control infection. In this regard cooperation
between TLR2 and TRIF may reflect the need for convergence of
the NF-kB and IRF pathways at the IFN-b promoter, with TLR2
being responsible mainly for NF-kB activation and TRIF being
instrumental for activation of IRF3 and IRF7. By employing the
strategy of PGN modification, L. monocytogenes can avoid immune
detection by TLR and evade the innate immune response, thus
enabling the infectious process to occur. It is important to recall
that pgdA orthologs are found in other pathogenic bacteria, such as
Streptococcus pneumoniae, Bacillus cereus, Bacillus anthracis and Helico-
bacter pylori, strongly suggesting that PGN N-deacetylation is a
general mechanism evolved by microbes to escape from pattern
recognition receptor-mediated immune recognition [39–42].
Materials and Methods
Bacterial strains and growth conditions
L. monocytogenes EGDe (BUG1600, ATCC BAA-679), L.
monocytogenes isogenic mutant DpgdA (BUG2288, ) and L.
monocytogenes DpgdA complemented strain (BUG2382) were grown
in brain heart infusion (BHI, Oxoid), aerobically at 37uC and
Construction of L. monocytogenes DpgdA complemented
A DNA fragment containing the pgdA gene (lmo0415) and its
promoter was generated by PCR using oligonucleotides lmo0415-
and lmo0415-2 (59-AAGGATCCTTATTTCACCATTCTT-
GAATCTG-39). The fragment was integrated into pCR-Blunt-
II-TOPO (Invitrogen) and the construct was verified by
sequencing. After digestion of the construct by BamHI, the
fragment was purified on agarose gel and cloned into the
integrative vector pPL2 , previously digested by BamHI,
constructing pOD98. The pOD98 was electroporated into DpgdA
at 2,500 V, 200 V and 25 mF. Transformants were selected at
37uC on BHI agar containing chloramphenicol (7 mg/mL). The
presence of the pgdA gene in the complemented strain was
confirmed by PCR using oligonucleotides lmo0415-1 and
Mice were used for obtaining peptone-elicited peritoneal
macrophages, resident peritoneal macrophages and bone mar-
row-derived macrophages. Animal experiments were performed in
accordance with protocols approved by the Animal Experimen-
tation Ethics Committee of the Institut Pasteur (permit #03-49)
and following Austrian law in accordance with protocols approved
by the Ethics Committee of the University of Veterinary Medicine,
Vienna (#GZ680 205/67-BrGt/2003).
Isolation and culture of murine peptone-elicited
peritoneal macrophages (PEM)
PEM were isolated from 7 to 10 week-old C57BL/6J and
genetically-matched tlr22/2, tlr32/2, tlr42/2, mal/tirap2/2, trif2/2,
irf32/2and irf72/2mice as previously described . The
percentage of macrophages was determined by flow cytometry
Listeria Induces IFN-b through TLR2 and TRIF
PLoS ONE | www.plosone.org6March 2012 | Volume 7 | Issue 3 | e33299
using CD11b (1:100, eBiosciences) and F4/80 (1:100, eBiosciences)
antibodies. More than 90% of the cells were macrophages. PEM
were seeded onto 6-well plates at a concentration of 26106cells per
well in DMEM (PAA) supplemented with 10% FCS, 10% L929
conditioned medium (LCM) and 1% penicillin-streptomycin or
RPMI-1640 (Gibco) supplemented with 10% FBS and 1%
Isolation of resident peritoneal macrophages
Resident macrophages were isolated from 6 to 8 week-old
C57BL/6J and genetically-matched tlr22/2, trif2/2mice by
washing the peritoneum twice with 10 mL DMEM (PAA)
supplemented with 10% FBS, 10% LCM and 1% penicillin-
streptomycin. Harvested cells were centrifuged at 300 g for
5 minutes and resuspended in complete medium. The percentage
of macrophages was determined by flow cytometry analysis as
above. Cells were seeded onto 6-well plates (Nunc) at a
concentration of 26106cells per well.
Isolation of bone marrow-derived macrophages
Tibia and femur from 6 to 8 week-old C57BL/6J and
genetically-matched tlr22/2, trif2/2mice were collected in ice
cold PBS. Bones were sterilized with 70% ethanol and flushed with
a 25-G needle using cold DMEM supplemented with 10% FCS,
10% LCM and 1% penicillin-streptomycin. Cells were seeded
onto 6-well plates (Nunc) at a concentration of 106cells per well
and incubated at 37uC with 5% CO2. After 4 days, complete
medium was added and cells were split at a ratio of 1:2. After 8
days, macrophages were fully differentiated.
Culture of human THP-1-derived macrophages and HEK-
blue type I IFN cells
Human acute monocytic leukemia THP-1 cells (ATCC
TIB202) were maintained in RPMI-1640 supplemented with
10% FBS and 1% penicillin-streptomycin. Cells were seeded onto
a 24-well plate at a concentration of 46105cells per well in
antibiotic-free media supplemented with 12.5 ng/mL phorbol
myristate acetate and incubated for 24 h at 37uC with 5% CO2.
Differentiation was determined to be successful upon formation of
a confluent adherent monolayer. HEK-blue type I IFN cells
(Invivogen) were grown in DMEM supplemented with 10% FBS
and 1% penicillin-streptomycin. Cells were seeded at a concen-
tration of 5.66104cells per well onto a 96-well plate.
Macrophage infection assays
For cytokine analysis, macrophages were infected with Listeria
strains at MOI 10:1, centrifuged at 300 g for 2 min and incubated at
37uC for 15 min. Following phagocytosis, monolayers were washed
twice followed by incubation in RPMI-1640 supplemented with
10% fetal bovine serum (FBS) and gentamicin (20 mg/mL).
Supernatants were collected at various time points, for detection of
IFN-b byELISA.For transcriptanalysis,macrophageswereinfected
with Listeria strains at MOI 20:1 and incubated at 37uC for 1 h to
allow phagocytosis. Monolayers were washed and incubated in
DMEM supplemented with 10% FCS and gentamicin (5 mg/mL).
After 2 h, medium was changed to DMEM supplemented with 10%
FCS and gentamicin (1 mg/mL). Cells were lysed at various time
points and RNA collected for qPCR analysis.
For inhibition of bacterial internalization, cell monolayers were
pretreated either for 2 h with 100 mM cytochalasin-D (Sigma-
Aldrich), or 30 min with 80 mM dynasore (Sigma-Aldrich) or
30 min with 100 mM chloroquine (Sigma-Aldrich) prior to
DNA isolation and transfection assays
Listeria were grown overnight in BHI at 37uC and cultures were
centrifuged at 8000 g for 5 min. Bacterial pellets were resuspended
in 75 mg/mL lysozyme and incubated at 37uC for 1 h. DNA was
then extracted using the DNeasy blood and tissue kit (Qiagen) and
quantified by spectrophotometry (Nanodrop). For transfection
assays, THP-1 macrophages were transfected with 200 ng/mL
DNA with 2% lipofectamine 2000 (Invitrogen) and incubated for
24 h. Following incubation, supernatants were collected for IFN-b
analysis. For pretreatment of DNA with DNase, DNase was added
at final concentration of 100 mg/mL for 45 min at 37uC.
Lysozyme digestion, quantification of nucleic acids
release and identification of Listeria PAMPs
Bacterial cultures were treated with 10 mg/mL lysozyme, a
concentration leading to lysis of DpgdA but not EGDe, and
incubated at 37uC and 200 rpm for 1 h. Following lysozyme
treatment, lysed bacterial cultures were centrifuged at 5000 rpm
during 10 min. Two types of experiments were performed on
supernatants. First, nucleic acid release was quantified. DNA was
purified using the Qiagen DNeasy blood and tissue kit omitting
lysis steps and quantified by spectrophotometry (Nanodrop). RNA
was purified using Qiagen RNeasy kit and quantified by
spectrophotometry (Nanodrop). Data shown are representatives
of at least three independent experiments. Second, 100 mL of each
supernatants were treated by DNase during 30 min at 37uC.
Enzymes were inactivated and treated- or untreated-supernatants
were transfected in PEM. 8 h after transfection, supernatants of
cells were recovered and the IFNb was quantified.
Detection of type I IFN by ELISA and HEK-blue type I IFN
Murine IFN-b production was detected in macrophage
supernatants by ELISA according to the manufacturer’s procedure
(PBL Biomedical Laboratories). For the HEK-blue type I IFN
assay, supernatant from THP-1 macrophage assays was collected
and 20 mL added onto HEK-blue type I IFN cells plated in 96-well
plates, which were incubated at 37uC overnight. Supernatant from
HEK-blue cells was collected and 40 mL added to 160 mL of
Quanti-blue reagent (Invivogen) for 20 min at 37uC. The
colorimetric reaction was measured at 625 nm on a plate reader.
Data was normalised against absorbance for the untreated cells
and plotted as relative fold increases. Data shown are represen-
tatives of at least three independent experiments.
Detection of IkB by immunoblot
PEM from WT or tlr22/2C57BL/6J mice were infected with
EGDe. Cells were lysed 0, 0.5, 1, 1.5, 2, 2.5, or 3 h post-infection.
IkB and tubulin were detected in lysates by immunoblotting using
anti-IkB (Santa Cruz, 1:100) and anti-a-tubulin (Sigma, 1:5000)
RNA isolation for quantitative real-time PCR
RNA preparation was performed using NucleoSpin RNA II kit
(Macherey-Nagel) according to the manufacturer’s instructions.
Quantitative real-time PCR was performed on a Mastercycler EP
realplex S (Eppendorf). Primers for HPRT (housekeeping gene
control), IFNb and IkBa mRNA expression were as follows:
HPRT reverse GAGGGTAGGCTGGCCTATTGGCT, IFNb
Listeria Induces IFN-b through TLR2 and TRIF
PLoS ONE | www.plosone.org 7March 2012 | Volume 7 | Issue 3 | e33299
forward 59-TCAGAATGAGTGGTGGTTGC-39, IFNb reverse
59-GACCTTTCAAATGCAGTAGATTCA-39; IkBa forward 59-
GCAATTTCTGGCTGGTGGG-39, IkBa reverse 59GATCC-
GCCAGGTGAAGGG-39. Data shown are representatives of at
least three independent experiments.
Results are expressed as means of at least three values, with
error bars representing standard deviations. Student’s t tests were
performed to determine statistical significance where*indicates
P,0.05,**indicates P,0.01 and***indicates P,0.0001.
Listeria in bone marrow macrophages. BMM from
C57BL/6J or trif2/2mice were infected with the parental EGDe
strain (black bars) or the DpgdA mutant (grey bars). After 4 h of
infection, IFN-b induction was measured by qRT-PCR. Data are
mean 6 SD (NS, non significant, n=3).
TRIF is not required for IFN-b response to
NF-kB. (A) PEM from WT C57BL/6J mice were infected with
EGDe. Cells were lysed 0, 0.5, 1, 1.5, 2, 2.5, or 3 h post-infection.
Activation of NF-kB was measured by determination of IkB
degradation relative to tubulin following immunodetection. (B)
PEM from tlr22/2mice were infected with EGDe. Cells were
lysed 0, 0.5, 1, 1.5, 2, 2.5, or 3 h post-infection. Activation of NF-
kB was measured by determination of IkB degradation relative to
tubulin following immunodetection.
TLR2 is required for optimal activation of
We thank members of the Cossart laboratory for helpful discussions.
Conceived and designed the experiments: OD PC LO TD. Performed the
experiments: CA SC SW CG RJ AMJ. Analyzed the data: OD PC LO TD
CA SC SW. Contributed reagents/materials/analysis tools: OD PC LO
TD. Wrote the paper: OD PC LO TD SC CA.
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