Innate Immune Sensing of Modified Vaccinia Virus
Ankara (MVA) Is Mediated by TLR2-TLR6, MDA-5 and the
Julie Delaloye1, Thierry Roger1, Quynh-Giao Steiner-Tardivel1, Didier Le Roy1, Marlies Knaup Reymond1,
Shizuo Akira2, Virginie Petrilli3, Carmen E. Gomez4, Beatriz Perdiguero4, Ju ¨rg Tschopp3, Giuseppe
Pantaleo5, Mariano Esteban4, Thierry Calandra1*
1Infectious Diseases Service, Department of Medicine, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland, 2Department of Host
Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan, 3Department of Biochemistry, University of Lausanne, Epalinges, Switzerland, 4Centro
Nacional de Biotecnologı ´a, CSIC, Ciudad Universitaria Cantoblanco, Madrid, Spain, 5Laboratory of AIDS Immunopathogenesis, Immunology and Allergology Service,
Department of Medicine, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Lausanne, Switzerland
Modified vaccinia virus Ankara (MVA) is an attenuated double-stranded DNA poxvirus currently developed as a vaccine
vector against HIV/AIDS. Profiling of the innate immune responses induced by MVA is essential for the design of vaccine
vectors and for anticipating potential adverse interactions between naturally acquired and vaccine-induced immune
responses. Here we report on innate immune sensing of MVA and cytokine responses in human THP-1 cells, primary human
macrophages and mouse bone marrow-derived macrophages (BMDMs). The innate immune responses elicited by MVA in
human macrophages were characterized by a robust chemokine production and a fairly weak pro-inflammatory cytokine
response. Analyses of the cytokine production profile of macrophages isolated from knockout mice deficient in Toll-like
receptors (TLRs) or in the adapter molecules MyD88 and TRIF revealed a critical role for TLR2, TLR6 and MyD88 in the
production of IFNb-independent chemokines. MVA induced a marked up-regulation of the expression of RIG-I like receptors
(RLR) and the IPS-1 adapter (also known as Cardif, MAVS or VISA). Reduced expression of RIG-I, MDA-5 and IPS-1 by shRNAs
indicated that sensing of MVA by RLR and production of IFNb and IFNb-dependent chemokines was controlled by the MDA-
5 and IPS-1 pathway in the macrophage. Crosstalk between TLR2-MyD88 and the NALP3 inflammasome was essential for
expression and processing of IL-1b. Transcription of the Il1b gene was markedly impaired in TLR22/2and MyD882/2BMDM,
whereas mature and secreted IL-1b was massively reduced in NALP32/2BMDMs or in human THP-1 macrophages with
reduced expression of NALP3, ASC or caspase-1 by shRNAs. Innate immune sensing of MVA and production of chemokines,
IFNb and IL-1b by macrophages is mediated by the TLR2-TLR6-MyD88, MDA-5-IPS-1 and NALP3 inflammasome pathways.
Delineation of the host response induced by MVA is critical for improving our understanding of poxvirus antiviral escape
mechanisms and for designing new MVA vaccine vectors with improved immunogenicity.
Citation: Delaloye J, Roger T, Steiner-Tardivel Q-G, Le Roy D, Knaup Reymond M, et al. (2009) Innate Immune Sensing of Modified Vaccinia Virus Ankara (MVA) Is
Mediated by TLR2-TLR6, MDA-5 and the NALP3 Inflammasome. PLoS Pathog 5(6): e1000480. doi:10.1371/journal.ppat.1000480
Editor: Grant McFadden, University of Florida, United States of America
Received November 12, 2008; Accepted May 21, 2009; Published June 19, 2009
Copyright: ? 2009 Delaloye 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: Research was conducted as part of the Poxvirus T Cell Vaccine Discovery Consortium (PTVDC) under the Collaboration for AIDS Vaccine Discovery with
support from the Bill & Melinda Gates Foundation. J.D. is supported by a grant from the Swiss National Science Foundation (number 313600-115680). 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: Thierry.Calandra@chuv.ch
Attenuated poxviruses are currently being developed as vaccines
vectors against various infectious diseases including HIV, malaria
and tuberculosis . Modified vaccinia virus Ankara (MVA) and
NYVAC are highly attenuated poxvirus strains due to multiple
deletions of viral genes and are replication-deficient in human
cells. MVA and NYVAC are immunogenic and safe and have
been shown to be excellent vaccine vectors for the expression of
foreign antigens. MVA is a leading vaccine candidate for delivery
of HIV genes with efficient induction of T-cell mediated immune
responses [1–3]. Profiling of the immune responses triggered by
poxvirus vaccine vectors is critical not only for optimal design of
vaccine vectors but also for anticipating potential harmful
interactions between naturally acquired or vaccine-induced
immune responses against the vaccine target. This is indeed an
important lesson learned from the adenovirus type 5 (Ad5) HIV
vaccine (MRKAd5) STEP trial. Pre-existing neutralizing antibod-
ies against the Ad5 vaccine vector were found to increase the
relative risk of HIV infection [4,5]. Hence the need for extensive
assessments of vaccine-induced innate and adaptive immune
responses to prevent unexpected adverse events.
Sensing of invasive pathogens by sentinel innate immune cells is
a fundamental feature of the host antimicrobial defense response.
Toll-like receptors (TLRs), retinoic acid-inducible gene-I (RIG-I)
like receptors (RLRs) and nucleotide-binding and oligomerization
domain (NOD)-like receptors (NLRs) have recently emerged as
central innate sensors of viruses . Virus sensing by TLR occurs
at the cell surface and in the endosomal compartment. At the cell
surface, TLR2 or TLR4 recognize either DNA (herpes viruses) or
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RNA viruses (respiratory syncitial, hepatitis C and measles viruses).
In the endosomal compartment, TLR7, TLR3 or TLR9 sense
single stranded (vesicular stomatitis virus, Sendai, West Nile and
influenza viruses) and double stranded (reovirus) RNA viruses, and
DNA viruses (herpes simplex viruses, cytomegalovirus), respec-
tively [7–13]. Two members of the cytosolic pattern recognition
RLR receptors, RIG-I (also known as DDX58) and melanoma
differentiation-associated gene 5 protein (MDA5) (also known as
helicard), have been shown to function as sensors of RNA viruses
[14–19]. RIG-I detects 59-triphosphate of ssRNAs and short
dsRNAs, while MDA5 preferentially recognizes long dsRNAs.
NALP3 (NLRP3 also known as cryopyrin) is a member of the
NLR family which have been involved in the sensing of both DNA
(adenovirus) and RNA (rotavirus, Sendai and influenza viruses)
viruses [20,21]. NALP3, ASC and pro-caspase 1 form a
multimeric cytosolic molecular complex known as the NALP3
inflammasome that controls the processing of the IL-1b cytokine
precursor pro-IL-1b into IL-1b . Sensing of viruses by TLRs,
RLRs and NLRs activates intracellular signalling pathways
resulting in the expression of pro-inflammatory cytokines and
type I interferons that then act on innate immune cells to limit
viral replication and promote the adaptive immune response.
Here we report that the TLR2-TLR6-MyD88, MDA-5-IPS-1
and NALP3 inflammasome pathways are the main innate sensors
of MVA in the macrophage and that they induce a cytokine
response profile characterized by a vigorous chemokine, IFNb and
IL-1b production. Beyond the dissection of the molecular bases of
MVA recognition by the innate immune system the present data
are likely to help design MVA vaccine vectors with improved
Innate immune responses elicited by MVA
The profile of innate immune responses elicited by MVA was
first examined by RT-PCR and ELISA in a mouse model of
poxvirus infection . MVA infection induced a robust innate
immune response in peritoneal cells, peritoneal lavage fluid,
splenocytes and splenocyte homogenates characterized by the
production of pro-inflammatory cytokines (TNF, IL-1b, IL-6, IL-
12p40), chemokines (IP-10/CXCL10, RANTES/CCL5, MCP-5/
CCL12, MIP-2/CXCL2) and type I interferon (IFNb) mRNA and
protein (Figure 1A and B and data not shown). Infection of human
whole blood with MVA also induced a vigorous innate immune
response characterized by an abundant production of chemokines
(IL-8/CXCL8, MIP-1a/CCL3 and IP-10) and less abundant
production of pro-inflammatory cytokines (TNF, IL-1b, IL-6)
(Figure 2). Interestingly, MVA was previously shown to down-
regulate IL-8 and IL-1b mRNA expression in human monocyte-
derived dendritic cells [24,25], suggesting that MVA infection may
induce the production of various patterns of cytokine depending
upon the cell-type studied.
Dissection of the molecular mechanisms of MVA-induced
innate immune responses was preformed in PMA-differentiated
human THP-1 macrophages and primary human macrophages.
Flow cytometry analyses performed with GFP-expressing MVA
(MOI 5) indicated that MVA rapidly infected THP-1 cells
(Figure 3A and B). More than 60% of cells became GFP positive
within 2 h followed by a progressive decline of GFP fluorescence
thereafter, which could result either from MVA-induced apoptosis
as observed in human HeLa and monocyte-derived dendritic cells
[24,25] or from the shutting down of protein synthesis through
activation of the PKR pathway by MVA . Indeed, the number
of apoptotic cells increased from 5% at 6 h to 35% at 24 h post-
infection as assessed by annexin V and propidium iodine staining
(data not shown).
The profile of cytokines and chemokines released by MVA-
infected THP-1 cells was analyzed with the Luminex technology.
Twenty four h after infection, 12 of the 30 mediators analyzed (see
Materials and Methods) were detectable in cell-culture supernatants.
Similarly to the results obtained with human whole blood (Figure 2)
and in agreement with a recent report by Lehmann et al. , MVA
induced the production of large quantities of chemokines (IL-8, MIP-
1a, MIP-1b/CCL4, MCP-1/CCL2, RANTES and IP-10). MVA
alsoinducedlarge amountsofIFNb and ofIL-1ra,but smallamounts
of pro-inflammatory cytokines (TNF, IL-1a, IL-1b, IL-6 and IL-
12p40) (Figure 3C and D). Kinetics and patterns of chemokines and
type I interferon mRNA expression were similar in MVA-stimulated
then also examined the production of cytokines and chemokines
induced by two other vaccinia virus (i.e. the attenuated NYVAC
strain and the virulent Western Reserve strain). When compared to
MVA, NYVAC induced low levels of IL-8, IL-1b and IFNb and no
TNF, IL-6, MIP-1a, RANTES or IP-10 (Figure S1). The virulent
Western Reserve strain of vaccinia virus was observed to also induce
low levels of IL-8 and IFNb in THP-1 cells, but no IL-1b, MIP-1a or
IP-10 (Figure S2 and data not shown).
Altogether, these results indicated that the innate immune
response induced by MVA in human macrophages was charac-
terized by a powerful chemokine production and a less abundant
production of pro-inflammatory cytokines probably related to the
attenuation of MVA . In contrast, the NYVAC and Western
reserve strains stimulated less powerful chemokine and cytokine
responses, that most likely reflect differences in the expression of
immunomodulatory genes in the genome of MVA, NYVAC and
Western Reserve [24,25].
TLR2, TLR6 and MyD88 are critical for IFNb-independent
chemokine production after MVA infection
TLRs have been shown to play an important role in the sensing
of viruses and in the initiation of the anti-viral host defense
Modified vaccinia virus Ankara (MVA) is a highly attenu-
ated, replication-deficient, poxvirus currently developed as
a vaccine vector against a broad spectrum of infectious
diseases including HIV, tuberculosis and malaria. It is well
known that robust activation of innate immunity is
essential to achieve an efficient vaccine response, and
that poxviruses have developed numerous strategies to
block the innate immune response. Yet, the precise
mechanisms underlying innate immune sensing of MVA
are poorly characterized. Toll-like receptors (TLR), RIG-I-like
receptors (RLR) and NOD-like receptors (NLR) are families
of membrane-bound and cytosolic sensors that detect the
presence of microbial products and initiate host innate
and adaptive immune responses. Here, we report the first
comprehensive study of MVA sensing by innate immune
cells, demonstrating that TLR2-TLR6-MyD88, MDA-5-IPS-1
and NALP3 inflammasome pathways play specific and
coordinated roles in regulating cytokine, chemokine and
interferon response to MVA poxvirus infection. Delineation
of the pathways involved in the sensing of MVA by the
host could help designing modified vectors with increased
immunogenicity, which would be of particular importance
since MVA is considered as a leading vaccine for HIV/AIDS
vaccine following the recent failure of an adenovirus-
mediated HIV vaccine trial.
Innate Immune Sensing of MVA
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response [29,30]. Analyses of the TLR repertoire used by the host
for sensing of MVA were conducted in bone marrow-derived
macrophages (BMDMs) isolated from TLR12/2, TLR22/2,
TLR42/2, TLR62/2, MyD882/2and TRIF2/2mice and the
read-out was the expression of IFN-independent chemokine MIP-
2 and of IFNb. MVA-induced MIP-2 production by BMDMs was
completely abrogated in TLR22/2, TLR62/2and MyD882/2
cells but not in TLR12/2, TLR42/2and TRIF2/2cells, which
produced amounts of MIP-2 similar to that of wild-type cells
(Figure 4A). In contrast, the amount of IFNb produced by
TLR22/2, TLR62/2and MyD882/2BMDMs was similar to
that of wild-type cells (Figure 4B), a finding consistent with the
notion that activation of the TLR2 pathway is not implicated in
the production of type I IFNs. Similar results were obtained with
THP-1 cells stably transduced with a lentiviral delivery system
expressing a short hairpin RNA (shRNA) targeting the expression
of the TLR2 gene (Figure S3). All together, these results indicated
that the activation of the TLR2-TLR6-MyD88 pathway was
required for the induction of IFNb-independent chemokines in
MVA-stimulated macrophages. Experiments conducted with
NYVAC and the Western Reserve strain of vaccinia virus
confirmed that TLR2 was required for IL-8 production by
THP-1 cells (Figure S1 and S2).
Endocytosis is required for IL-1b and IFNb production
Vaccinia virus penetrates into target cells either by endocytosis
or by membrane fusion in a low pH-independent manner . To
determine the contribution of endocytosis to MVA-induced
intracellular signalling and cytokine production, THP-1 cells were
treated with cytochalasine D, an actin-depolymerizing drug that
blocks the endocytotic trafficking, or with chloroquine, a
lysosomotropic weak base to neutralize the acidic environment
of endocytic vesicles. IL-1b and to a lesser extend IFNb
production were inhibited by cytochalasine D and chloroquine
treatment. The inhibition was not related to drug toxicity because
chloroquine did not affect IL-8 production and cell viability
(Figure 5 and data not shown). The reason why the inhibition of
cytokine production (particularly IFNb) was only partial after
treatment with the inhibitors remains uncertain. The data suggest
that additional non-endocytic pathways may play a role in the
production of IFNb. In agreement with a key role for membrane-
bound TLR2 for IL-8 induction, the production of IL-8 was not
reduced after cytochalasine D or chloroquine treatment (Figure 5).
UV treatment of MVA causing a nearly complete (i.e. 90%)
inhibition of the expression of the early C6L gene (data not shown)
did not affect IL-1b, IL-8 and IFNb production (Figure 5).
Although one cannot completely rule out a contribution of residual
Figure 1. MVA stimulates cytokine, chemokine and IFNb production in vivo. BALB/c mice were injected i.p. with MVA (107PFU). Peritoneal
cells (A) and peritoneal lavage fluid (B) were collected 12 h after infection as described in Materials and Methods. TNF, IL-1b, IL-12p40, IP-10, RANTES
and IFNb mRNA contents of peritoneal cells were analyzed by RT-PCR (A). Results are expressed as the ratio of cytokines, chemokines or IFNb mRNA
levels to that of HPRT. AU: arbitrary units. Cytokine concentrations in peritoneal lavage fluid were measured by ELISA (B). Data are means6SD of
triplicate samples from one experiment comprising three mice per experimental condition and are representative of two independent experiments.
p,0.05 for all conditions when comparing PBS versus MVA.
Innate Immune Sensing of MVA
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viral protein synthesis, these observations support the view that
induction of cytokines by MVA is most likely independent of viral
gene synthesis [32–34]. Overall, endocytosis of MVA was required
for IL-1b and IFNb release suggesting a role for intracellular
pattern recognition receptors in the production of these cytokines.
MVA is sensed by MDA-5 and not by RIG-I
The RLR family of cytosolic pattern recognition receptors has
been implicated in the sensing of RNA viruses , but very little
is known about their role in host response to DNA viruses.
Extending the observations by Guerra et al. who noted an
increased expression of RIG-I and MDA-5 mRNA in human
dendritic cells infected with MVA , we observed that MVA
caused a time-dependent increase in RIG-I, MDA-5 and IPS-1
mRNA and protein expression in THP-1 cells (Figure 6A and B).
RIG-I and MDA-5 mRNAs rose within 3 h of infection and
remained elevated for up to 24 h (Figure 6A). In vivo, MVA up-
regulated RIG-I and MDA-5 mRNA levels in peritoneal cells and
splenocytes (Figure S4). When compared to MVA, NYVAC
induced lower levels of MDA-5 and, to a lesser extent, RIG-I and
IPS-1 mRNA and protein expressions (Figure S1 and data not
shown). Using shRIG-I, shMDA-5 and shIPS-1 THP-1 cells
(Figure S5), we then examined whether RIG-I and MDA-5 were
involved in MVA-induced IFNb production. IFNb and IP-10
mRNA and protein levels were markedly reduced in shMDA-5
and shIPS-1 cells, but not in shRIG-I cells. By contrast, the time-
course and magnitude of the IL-8 and IL-1b production was
similar in shMDA-5, shIPS-1, shRIG-I and control THP-1 cells
(Figure 7A and B). Sensing of MVA by the MDA-5/IPS-1
pathway is therefore critical for the production of IFNb and IFNb-
dependent chemokines in macrophages. In line with these data,
the production of IFNb, but not of IL-8, was also dependent on
the MDA-5/IPS-1 pathway in cells infected with NYVAC and the
Western Reserve strain of vaccinia virus (Figure S1 and S2).
Crosstalk between TLR2-MyD88 and the NALP3
inflammasome for IL-1b expression and processing
IL-1b is a key cytokine of antimicrobial host defenses, whose
expression is regulated at a transcriptional and post-transcriptional
level . IL-1b is likely to play an important role during poxvirus
infection, as suggested by the fact that poxviruses encode for IL-1b
decoy receptor and disrupt intracellular IL-1 receptor signalling
[37,38]. We therefore examined whether activation of the TLR2-
MyD88 pathway was implicated in the activation of the IL1b gene.
As shown in Figure 8A, up-regulation of IL-1b mRNA was
markedly impaired in TLR22/2and MyD882/2BMDMs
infected with MVA, indicating that activation of the TLR2-
MyD88 signalling pathway is critical for transcription of the IL1b
gene during MVA infection. Secretion of mature IL-1b p17 in
response to endogenous and exogenous danger signals requires the
cleavage of the inactive pro-IL-1b precursor by the cysteine
protease caspase-1. Conversion of pro-caspase-1 into caspase-1 is
tightly regulated by the NALP3 inflammasome composed of
NALP3, ASC and pro-caspase-1 . To examine the contribu-
tion of the NALP3 inflammasome in the production of IL-1b
triggered by MVA, we analyzed the expression of pro-IL-1b and
IL-1b p17 in THP-1 cells deficient in NALP3, ASC or caspase-1
. Knocking down of either one of the three components of the
NALP3 inflammasome (i.e. NALP3, ASC or caspase-1) was
associated with a massive reduction of mature and secreted IL-1b
(Figure 8B and C). Similar results were obtained in THP-1 cells
infected with NYVAC (Figure S1) and in NALP32/2BMDMs
infected with MVA (Figure 8D and E). Of note, in THP-1 cells
and in BMDMs the expression of pro-IL-1b was unaffected by the
Figure 2. TNF, IL-1b, IL-6, IL-8, MIP-1a and IP-10 release by human whole blood exposed to MVA. Whole blood from 3 healthy volunteers
(#1, 2 and 3) was incubated for 24 h with (+) or without (2) MVA (MOI 1) in triplicates. Cell-free supernatants were collected to quantify the
concentrations of TNF, IL-1b, IL-6, IL-8, MIP-1a and IP-10. Data are means6SD of triplicate samples from one experiment. MVA significantly increased
cytokine production (p,0.05 for all conditions).
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Figure 3. MVA induces the production of cytokines, chemokines and IFNb by human macrophages. Human THP-1 cells (A–E) and
primary human macrophages (F) were infected with GFP-positive (A, B) or wild-type (C–F) MVA (MOI 5). Expression of viral-derived GFP protein by
THP-1 cells analyzed by flow cytometry (A, B). Cytokines, chemokines and IFNb production by THP-1 cells stimulated for 24 h with MVA as assessed by
the Luminex technology (C) or by ELISA (D). IL-8 (CXCL8), MIP-1a (CCL3), RANTES (CCL5), IP-10 (CXCL10) and IFNb mRNA levels were analyzed by RT-
PCR and results expressed as the ratio of chemokines or IFNb to HPRT mRNA levels. AU: arbitrary units (E, F). Data are means6SD of duplicate (C) or
triplicate (D to F) samples from one experiment and are representative of one (C) to three (D to F) independent experiments. p,0.05 for all conditions
(D to F).
Innate Immune Sensing of MVA
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absence of either NALP3, ASC or caspase-1 clearly indicating that
NALP3 inflammasome does not itself regulate the transcriptional
and translation control of the IL-1b precursor. The NALP3
inflammasome was also dispensable for activation of the IRF3
transcription factor and IFNb secretion (Figure S6). Altogether,
these data demonstrate that IL-1b production after MVA infection
Figure 4. TLR2, TLR6 and MyD88 are critical for IFNb-independent chemokine production after MVA infection. MIP-2 (A) and IFNb (B)
produced by wild-type, TLR12/2, TLR22/2, TLR42/2, TLR62/2, MyD882/2and TRIF2/2bone marrow-derived macrophages infected with MVA (MOI 5
and 20) or stimulated with lipopolysaccharide (LPS, 100 ng/ml), Pam2CSK4(P2CSK4, 10 mg/ml), Pam3CSK4(P3CSK4, 10 mg/ml) for 24 h. Data are
means6SD of triplicate samples from one experiment and are representative of 2 to 4 experiments.
Figure 5. Endocytosis is required for IL-1b and IFNb production after MVA infection. THP-1 cells were preincubated for 1 h with or without
cytochalasin (2 mM) or chloroquine (100 mM) prior to exposure to MVA or UV-treated MVA (MOI 20). Cell-culture supernatants were harvested after
6 h (IL-1b) or 24 h (IFNb and IL-8) and cytokine concentrations were measured by ELISA. Data are means6SD of triplicate samples from one
experiment and are representative of two independent experiments.
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requires a crosstalk between TLR2-MyD88 (initiation of the
transcription and translational of IL-1b) and the NALP3
inflammasome (processing of pro-IL-1b into mature IL-1b).
MVA activates the NF-kB, ERK1/2, JNK, IRF3, IRF7 and
STAT-1 signalling pathways
Poxviruses have been reported to activate the NF-kB, ERK1/2
and JNK pathways in epithelial and fibroblastic cell lines [40–43]
and IRF3 and IRF7 in dendritic cells [24,25]. Having identified
the pathogen recognition receptors implicated in macrophage
response to MVA (TLR2-TLR6, MDA-5 and NALP3), we next
examined which downstream signalling pathways are activated for
the expression of cytokines, chemokines and type I IFNs. Kinetics
studies of NF-kB, ERK1/2 and JNK MAP kinases and IRFs
activation were performed in THP-1 cells (Figure 9A). Electro-
phoretic mobility shift assay revealed that NF-kB nuclear content
peaked 3 h after MVA infection. Phosphorylation of the ERK1/2
and JNK MAP kinases was between 1 and 6 h after infection.
IRF3, which is essential for transcription of the IFNB gene, was
detected 3 h after infection, peaked at 6 h and rapidly decreased
thereafter. IRF7 was detected 3 h after infection and levels
remained unchanged for 24 h. Phosphorylation of signal trans-
ducer and activator of transcription 1 (STAT-1), a critical target of
IFNb signalling required for the transcriptional activation of
IFNb-dependent genes, was first detected 3 h post-infection and
gradually increased until 24 h (Figure 9A). The functional
significance of the increased binding activity of NF-kB and
phosphorylation of the IRF3 was confirmed by showing that MVA
increased the transcriptional activities of multimeric-kB and IRF3-
dependent-IFNb promoter luciferase reporter vectors in transient-
ly transfected THP-1 cells (Figure 9B and C). Confirming the
importance of NF-kB and ERK1/2 in mediating innate immune
response to MVA infection, pre-incubation of THP-1 cells with
drugs (i.e. NEMO and U0126, see Materials and Methods) selectively
inhibiting the NF-kB and ERK-1/2 signalling pathways impaired,
albeit to a different extent, IL-1b (70% and 65% inhibition), IL-8
(75% and 72% inhibition) and IFNb (28% and 42% inhibition)
mRNA expression (p,0.05 for all conditions). Therefore,
consistent with the fact that several pattern recognition receptors
are engaged in the sensing of MVA by the innate immune system,
multiple intracellular signalling pathways, including NF-kB, MAP
kinases and IRFs were found to be activated upon infection of
THP-1 macrophages with MVA. Of note, NYVAC induced very
weak induction of intracellular signalling (i.e. NF-kB, ERK-1/2,
IRF3 and STAT-1) and low levels of cytokines and IFNb when
compared with MVA (Figure S1) which is likely due to the
expression of different patterns of immunomodulatory genes by
these two poxviruses [24,25].
Analyses of pattern recognition receptors engagement by
poxviruses are essential for improving our understanding of the
pathogenesis of this important class of DNA viruses and for
designing new viral vaccine vectors with improved immunogenic-
ity. Dissection of the molecular bases of innate immune responses
elicited by the attenuated poxvirus MVA strain in human
macrophages revealed a critical role for TLR2-TLR6-MyD88,
MDA-5-IPS-1 and NALP3 inflammasome pathways in the
production of chemokines, IFNb and IL-1b. These observations
provide novel information on MVA recognition by sentinel innate
immune cells and highlight the existence of potential differences
between attenuated and non-attenuated poxviruses in the
engagement of or recognition by innate sensors.
Up to now the retinoic acid-inducible gene-I-like receptors
(RLR) RIG-I and MDA-5 had been viewed as master cytosolic
sensors of RNA viruses . However, recent observations
suggested a role for the RLR pathway in the recognition of
DNA viruses. Mouse embryo fibroblasts deficient in IPS-1
displayed reduced induction of IFNb in response to MVA lacking
the E3 protein . Adenovirus and HSV1 have also been shown
to replicate at much higher titers in RIG-I mutant than in RIG-I
wild-type human hepatoma cell lines . Moreover, microarray
analyses revealed that RIG-I and MDA-5 expression was
upregulated in human monocyte-derived dendritic cells infected
with MVA . Here we also showed that MVA caused a strong
up-regulation of RIG-I, MDA-5 and IPS-1, yet only MDA-5 and
IPS-1 were found to mediate MVA-induced IFNb and IFNb-
dependent chemokine production by macrophages (Figure 10). As
anticipated, transcriptional activation of IFNb and IFNb-dependent
chemokine genes was associated with the activation of IRF3 and
IRF7 and STAT-1. To the best of our knowledge this is the first
demonstration of a direct role for MDA-5 in innate sensing of a
DNA virus. Moreover, the MDA-5/IPS-1 pathway was also
implicated in the production of IFNb by macrophages infected
with the NYVAC and the Western Reserve strains of vaccinia
virus (Figure S1 and S2).
RIG-I has been shown to be involved in the induction of TNF
and type I IFN by myxoma poxvirus in human macrophages .
Figure 6. MVA up-regulates the expression of RIG-I, MDA-5 and IPS-1 mRNAs and proteins. RIG-I, MDA-5 and IPS-1 mRNA and protein
expression by RT–PCR (A) and Western blot (B). THP-1 cells were infected with MVA (MOI 5) for the indicated time. Results are expressed as the ratio
of RIG-I, MDA-5 or IPS-1 mRNA levels to that of HPRT. Data are means6SD of triplicate samples from one experiment and are representative of three
independent experiments. AU: arbitrary units. *p,0.05.
Innate Immune Sensing of MVA
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Yet, silencing of MDA-5 was associated with a small (about 25%)
but clear reduction of macrophages response to myxoma virus
suggesting that both RIG-I and MDA-5 were implicated, albeit to
various degree, in innate immune response to myxoma virus. The
nature of the component(s) of DNA viruses activating the RLR
pathway remains to be identified. Obvious candidate molecules
include, envelope or core proteins, early mRNA and DNA itself.
Unless RLR engagement is used primarily to the virus own
benefit, it is likely that poxviruses have developed antiviral escape
strategies interfering with the host RLR antiviral defense pathway.
In line with this assumption, the dsRNA binding protein E3 of
vaccinia virus has been reported to inhibit IPS-1 signaling, IRF3
phosphorylation, cytokine and IFNb production [47–49]. Should
inhibitors of the RLR pathway be identified in the MVA genome,
gene deletion might provide an opportunity to generate new MVA
vaccine vectors with increased immunogenicity.
In addition to RLR, profiling of the cytokine response induced
by MVA in the macrophage revealed a key role for the
Figure 7. MVA is sensed by MDA-5 and not by RIG-I. THP-1 cells stably transduced with control, MDA-5, RIG-I or IPS-1 shRNAs were infected
with MVA (MOI 5 unless specified otherwise) for the indicated time. IFNb, IP-10, IL-8 and IL-1b mRNA and protein expression by RT-PCR and ELISA (A–
B). Results are expressed as the ratio of IFNb, IP-10, IL-8 or IL-1b mRNA levels to that of HPRT. Data are means6SD of triplicate samples from one
experiment and are representative of four independent experiments. AU: arbitrary units. Concentrations of IFNb and IL-8 in cell-culture supernatants
were measured 24 h after stimulation. shMDA-5 and shIPS-1 THP-1 cells produced significantly less IFNb and IP-10 mRNA and protein than control
cells as measured 24 h post-infection (A and B) (p,0.05).
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heterodimeric TLR2-TLR6 complex and the adapter protein
MyD88 in the production of IFNb-independent chemokines (such
as IL-8, MIP-1a, MIP-1b and MIP-2) (Figure 10). Innate immune
recognition of the vaccinia virus has also been shown to depend on
TLR2 and MyD88 . The present observation is one of the few
examples of viral recognition mediated by TLR2 heterodimers.
Recognition of human cytomegalovirus has been shown to be
mediated by a TLR2-TLR1 heterocomplex and that of hepatitis C
virus by either TLR2-TLR1 or TLR2-TLR6 [50,51]. The facts
that TLR2 is expressed at the cell surface and that the inhibition of
endocytosis or UV-irradiation of MVA did not affect IL-8
production by macrophage suggest that a component of the
MVA envelope or a core protein is responsible for the activation of
the TLR2-TLR6-MyD88 pathway. However, the nature of the
viral component likely to serve as ligands for these TLR2-TLR1/
TLR6 heterodimers has so far remained elusive.
Other TLRs have also been implicated as innate sensors of
poxviruses. Ectromelia virus, the causative agent of mousepox, was
shown to be recognized by mouse dendritic cells in TLR9
dependent manner . In contrast, responses of dendritic cells to
MVA was both TLR9-dependent (up-regulation of CD40) and
TLR9-independent (up-regulation of CD69 and production of
IFNa and IL-6) [33,52]. Although we did not perform experiments
with TLR9-deficient macrophages in the present study, the data
obtained with MyD88 deficient cells clearly rule out the
implication of TLR9 in MVA-induced IFNb and IFNb-dependent
chemokines. However, we cannot exclude the involvement of
TLR9 in the production of IFNb-independent chemokines.
Finally, in a mouse model activation of TLR3 contributed to the
pathogenesis of Western Reserve vaccinia virus . In contrast,
experiments conducted with TRIF-deficient macrophages clearly
showed that the production of chemokines and IFNb induced by
MVA was TLR3-independent in the present study. Taken
together these observations demonstrate that TLRs may exert a
two-sided role in poxvirus infections acting on the one hand as key
initiators of the host anti-poxvirus defense response and on the
other hand as important mediators of viral pathogenicity and
The other important intracellular innate immune sensor of
microbial products and endogenous molecules is the NALP3
inflammasome that controls the processing and maturation of the
cytokines IL-1b and IL-18 . Here we show that MVA is a
potent activator of the NALP3 inflammasome and of IL-1b release
by macrophages. IL-1b and IL-18 are key mediators of the host
antimicrobial defense response and several lines of evidence
suggest that these cytokines are likely to play an important role in
host defenses against poxvirus infections. For example, the B15R
gene of the vaccinia virus encodes an IL-1b decoy receptor
blocking the activity of IL-1b and IL-18 and inactivation of B15R
gene reduces the virulence of the vaccinia virus [38,54].
Furthermore, poxviruses release IL-18 binding proteins inhibiting
IL-18 activity and vaccinia viruses A46R, A52R, N1L and, K1L
gene products have been shown to disrupt the IL-1 receptor
intracellular signaling pathway at multiple levels [37,55]. Inter-
estingly, we observed that MVA stimulated the release of large
amounts of the IL-1 receptor antagonist by macrophages
(Figure 3C) adding further support to the view that IL-1 is an
important target of the poxvirus antiviral escape strategy. Finally,
consistent with the notion that the NALP3 inflammasome plays an
important role in host defenses against poxviruses, several
Figure 8. Crosstalk between TLR2-MyD88 and the NALP3 inflammasome for IL-1b expression and processing. (A) Wild-type, TLR22/2
and MyD882/2BMDMs were primed overnight with ultra-pure LPS (100 ng/ml) and infected with MVA (MOI 5). IL-1b mRNA expression was
quantified by RT-PCR (p,0.05 for TLR22/2or MyD882/2vs. wild-type BMDMs). THP-1 cells stably transduced with control, NALP3, ASC and caspase 1
(casp1) shRNAs were infected with MVA (MOI 5 unless specified otherwise) for the indicated time (B–C). (B) Western blots of intracellular pro-IL-1b and
secreted IL-1b p17. (C) IL-1b concentrations measured by ELISA in cell-culture supernatants collected 24 h after infection (p,0.05 for cells transduced
with NALP3, ASC and casp1 shRNAs vs. control shRNA). LPS-primed wild-type and NALP32/2BMDMs were infected with MVA (MOI 5 in D) for 6 h (D–
E). (D) Western blots of intracellular pro-IL-1b and secreted IL-1b p17. (E) IL-1b concentrations measured by ELISA in cell-culture supernatants
collected 24 h after infection. Results are expressed as the ratio of IL-1b mRNA levels to that of HPRT. Data are means6SD of triplicate samples from
one experiment and are representative of two independent experiments (p,0.05 for NALP32/2vs. wild-type BMDMs).
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inhibitors of caspase-1 and ASC, like CrmA (cowpox virus),
M13L-PYD (myxoma virus) and PYD-only (shope fibroma-virus)
have been identified in the genomes of several poxviruses [56–58].
Crosstalks between TLRs and NLRs have been demonstrated to
occur in the course of bacterial infections, such as between TLR5
and the IPAF inflammasome after exposure to flagellated bacteria
or the flagellin protein itself [59–61]. To the best of our knowledge,
however, the present data provide the first demonstration of a
crosstalk between the TLR and NLR pathways in the context of a
viral infection (Figure 10). While TLR2 and MyD88 were necessary
to induce IL-1b mRNA expression (Figure 8A), the NALP3
inflammasome was absolutely required for the processing of pro-
IL-1b and IL-1b secretion (Figure 8B and C). Dual activation
pathways coupling MVA recognition to IL-1b may provide the host
with an increased capacity of fine tuning of its cytokine response.
In summary, the present data show that the TLR2-TLR6-
MyD88, MDA-5-IPS-1 and NALP3 inflammasome pathways
exert both specific and coordinated functions in the sensing of
MVA infection and in the regulation of cytokine, chemokine and
IFNb responses (Figure 10). After the unfortunate failure of the
adenovirus type 5 HIV vaccine STEP trial due to issues related to
natural immunity against this virus, the attenuated MVA and
NYVAC strains of poxvirus have become attractive vaccine
vectors against HIV/AIDS. Arguments supporting the use of
MVA and NYVAC as vaccine vectors include excellent immuno-
genicity and safety profiles and limited pre-existing immunity to
poxvirus in the population at risk of HIV infection due to the
abandon of vaccine campaigns after the eradication of smallpox in
the 1970s. The present findings are therefore likely to provide
important information relevant to the study of the pathogenesis of
poxvirus infections, the understanding of antiviral escape mech-
anisms of poxvirus and may help to design new vaccine vectors
with increased immunogenicity.
Materials and Methods
All animal procedures were approved by the Office Ve ´te ´rinaire
du Canton de Vaud (authorizations nu 876.5, 876.6, 877.5 and
877.6) and performed according to our institution guidelines for
Eight to ten-week-old female BALB/c and C57BL/6 mice were
purchased from Charles River Laboratories (L’Arbresle, France)
and were acclimatized for at least one week before experimenta-
tion. MyD882/2, TRIF2/2, TLR12/2, TLR22/2, TLR42/2,
TLR62/2and NALP32/2C57BL/6 mice have been described
Figure 9. MVA activates the NF-kB, ERK1/2, JNK, IRF3, IRF7 and STAT-1 signalling pathways. Electrophoretic mobility shift assay of NF-kB
DNA binding activity and Western blots of phosphorylated ERK1/2 (P-ERK1/2), JNK (P-JNK), IRF3 (P-IRF3) and STAT-1 (P-STAT-1) and total ERK1/2, JNK
and IRF7 (A). Nuclear (NF-kB) and cytosolic (ERK1/2, JNK, IRF3, IRF7, STAT-1 and tubulin) extracts were prepared from THP-1 cells infected with MVA
(MOI 5) for the indicated time. Results are representative of three independent experiments. The retarded complex detected by EMSA was dose-
dependently inhibited by cold wild-type but not mutant NF-kB oligonucleotide, and supershifted using anti-p65 antibody (data not shown). NF-kB-
(B) and IRF3- (C) mediated transcriptional activities measured in THP-1 cells transiently transfected with trimeric kB sites or IRF3-dependent IFNb
promoter luciferase reporter vectors and infected with MVA (MOI 5 and 20) for 18 h. Results are expressed as the ratio of luciferase activity to Renilla
luciferase activity. Data are means6SD of triplicate samples from one experiment and are representative of four independent experiments. p=0.05,
0.02, 0.04 and 0.02 for MVA-infected (MOI 5 and 20) vs. control cells.
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previously [62–68]. Mice were bred and housed in specific
pathogen free conditions.
Cells and reagents
The human monocytic THP-1 cell line (American Type Culture
Collection, Manassas, VA) was cultured in RPMI 1640 medium
containing2 mM L-glutamine,
100 IU/ml of penicillin, 100 mg/ml of streptomycin (all from
Invitrogen, San Diego, CA) and 10% heat-inactivated FCS
(Sigma-Aldrich, St. Louis, MO). THP-1 cells differentiated into
macrophages by treatment with 0.5 mM phorbol 12-myristate 13-
acetate (PMA, Sigma-Aldrich) for 24 h were used in all
experiments except those for reporter gene analyses. THP-1 cells
stably expressing control, NALP3, caspase-1 and ASC shRNA
have been described previously [69,70]. THP-1 cells expressing
TLR2, IPS-1, MDA-5 and RIG-I shRNA were generated using
lentiviruses expressing hairpins directed against TLR2, IPS-1 and
MDA-5 (5 for TLR2, 5 for IPS-1, 2 for MDA-5 and 5 for RIG-I)
produced with the second-generation pMD2-VSVG and pCMV-
R8.91 packaging plasmids as described previously and cultured in
the presence of 5 mg/ml puromycin . The sequence of the
hairpins selected that gave the best targeting of TLR2, IPS-1,
GAAAAG, CAAGTTGCCAACTAGCTCAAA, CCAACAAA-
GAAAAG, respectively. Levels of expression of targeted genes
were analyzed by real-time PCR using specific oligonucleotides
50 mM 2-mercaptoethanol,
(Table S1) and the most efficiently silenced THP-1 subsets were
selected for further studies (i.e. cell lines #1 in Figure S2).
Peripheral blood mononuclear cellsfrom healthy donors (recruited
by the Blood Center, Lausanne, Switzerland) were purified by Ficoll-
Hypaque density gradient (GE Healthcare, Uppsala, Sweden).
Macrophages were obtained by culturing adherent PBMCs cells for
6 days in RPMI 1640 with Glutamax. Bone marrow-derived
TLR22/2, TLR42/2, TLR62/2, MyD882/2and TRIF2/2mice
were cultured for 7 days in IMDM (Invitrogen) containing 50 mM 2-
mercaptoethanol and monocyte-colony stimulating factor to obtain
BMDMs. All media were supplemented with 10% FCS, 100 IU/ml
of penicillin and 100 mg/ml of streptomycin. In selected experiments,
cells were stimulated with 100 ng/ml Salmonella minnesota ultra pure
LPS (List Biologicals Laboratories, Campbell, CA), 10 mg/ml
polyinosine-polycytidylic acid (poly(I:C), Invivogen, San Diego,
CA), 1–10 mg/ml S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cystei-
(Pam2CSK4) or N-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-
(Pam3CSK4) lipopeptides (EMC microcollections, Tuebingen, Ger-
San Diego, CA), 2 mM cytochalasine D, 100 mM chloroquine
(Sigma-Aldrich), 10 mM SB203580 (p38 inhibitor), 10 mM U0126
(MEK1/2 inhibitor) or 50 mg/ml NEMO-binding domain binding
peptide (IkB kinase inhibitor) (Calbiochem-Novabiochem, Notting-
from wild-type, TLR12/2,
Figure 10. Pathways activated by MVA in the macrophage. Infection of macrophages with MVA stimulates the TLR2-TLR6-MyD88, MDA-5/IPS-
1 and NALP3 inflammasome pathways leading to the activation of NF-kB, ERK-1/2, JNK, IRF3, IRF7 and STAT-1 that are involved in the transcriptional
activation of genes encoding for cytokines, chemokines and type I IFN. At the cell surface, MVA is sensed by the TLR2-TLR6 heterodimer that induces
the production of IFNb-independent chemokines (IL-8, MIP-1 and MIP-2) (1) and pro-IL-1b (2). Upon virus entry into the cell, cytosolic MVA or MVA-
derived viral components (possibly envelope or core proteins, early mRNA or DNA) activate the MDA-5-IPS-1 pathway to release IFNb (3) and
subsequent induction of IFNb-dependent chemokines (such as RANTES, IP-10) following activation of the type I IFN receptor (4). Finally, MVA
infection leads to the activation of the NALP3 inflammasome (composed of NALP3, ASC and pro-caspase 1) enabling caspase-1 processing, pro-IL-1b
maturation and IL-1b secretion (5). For simplicity, the same diagram for MVA is shown outside and inside the cell.
Innate Immune Sensing of MVA
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MVA, NYVAC and WR production, in vitro and in vivo
models of infection
MVA and NYVAC were cultured in chicken embryo fibroblasts
and WRin HeLa cells. Viruses were purified by two sucrose cushions
and titrated on BHK-21 and BSC-40 cells as previously described
[24,72]. Cells were infected with MVA, NYVAC or WR at various
multiplicities of infection (MOI 1, 5 or 20 pfu/cell). After 1 h of
contact with cells,the virusinoculumwasremovedandfresh medium
added to the cultures. Cell-culture supernatants and cells were
collected at different time points after infection and processed for flow
cytometry, Luminex technology, ELISA, RNA extraction, and
Western blot analyses. In selected experiments, MVA suspension
(0.2 ml in 24-well plates laid on ice) was irradiated by a 15-min
exposure to a 365-nm UV bulb at a distance of 4 cm. UV-irradiation
caused a 90% inhibition of the expression of C6L early gene as
determined by RT-PCR using oligonucleotides (59-39 sense and
antisense at position 219541/219503 and 219071/219090 in
GATTCGTTTTCTTTAGAG and CGGGATCCTTACTTGT-
fAAATATC. MVA did not propagate in THP-1 cells as demon-
strated by the absence of infective viral particles in cell-culture
supernatants collected 24 h after infection (data not shown).
For whole blood assay, 100 ml of heparinized whole blood
collected from 3 healthy volunteers were diluted 5-fold in RPMI
1640 medium containing MVA (MOI 1) and incubated for 24 h at
37uC in the presence of 5% CO2. Samples were centrifuged, and
cell-free supernatants were stored at 280uC until cytokine
measurement. For in vivo studies, 26107PFU of MVA in 1 ml
phosphate-buffered saline (PBS) were injected intraperitoneally
into BALB/c mice. After 12 h, a peritoneal lavage was performed.
The supernatant obtained after centrifugation of the lavage fluid
was collected for cytokine measurement by ELISA whereas the cell
pellet was processed for gene expression analysis by RT-PCR.
Spleens were collected from the same animals to quantify cytokine
protein and mRNA expression levels.
To followcellinfection, THP-1cellswereinfected (MOI5)witha
GFP-expressing mutant MVA, whereas all other experiments used
wild-type MVA. The percentage of GFP-positive THP-1 cells was
measured 0, 2, 4, 6, 12 and 24 h after infection. MVA-induced cell
apoptosis was determined 6 h and 24 h post-infection using the
Annexin-V FITC apoptosis detection kit according to manufactur-
er’s recommendations (BD Biosciences, Erembodegem, Belgium).
Acquisition and analysiswereperformed usinga FACS Calibur (BD
Biosciences) and FlowJo 8.5.3 software (FlowJow, Ashland, OR).
Measurement of cytokine production
A screening of mediators produced by MVA-infected THP-1
cells was performed with the human cytokine Bioplex assay (Bio-
Rad, Hercules, CA) using the Luminex technology (Luminex
Corporation, Austin, TX) available at the Cardiomet Mouse
Metabolic Evaluation Facility, Center for Integrative Genomics,
University of Lausanne, Lausanne, Switzerland. Thirty mediators
were tested: TNFa, IL-1a, IL-1ra, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-
7, IL-8, IL-10, IL-12p40, IL-12p70, IL-13, IL-15, IL-17, IFNc,
RANTES, IP-10, MIP-1a, MIP-1b, MCP-1, eotaxin, fractalkine,
TGFa, EGF, VEGF, GM-CSF, G-CSF and sCD40L. The
concentrations of human IL-1b (Bender MedSystems, Vienna,
Austria), IL-8, (BD Biosciences), IP-10, MIP-1a (R&D) and IFNb
(PBL Biomedical Laboratories, Picataway, NJ) in whole blood assay
and cell-culture supernatants were measured by ELISA. TNF and
IL-6 concentrations were measured by bioassay as described
elsewhere . Mouse IL-1b, MIP-2 (R&D) and IFNb were
quantified by ELISA (Biomedical Laboratories, Picataway, NJ).
RNA analysis by quantitative real-time polymerase chain
Total RNA was isolated from THP-1 cell lines, human
monocytes/macrophages, peritoneal cells and splenocytes using
the RNeasy kit (Qiagen, Hombrechtikon, Switzerland). Reverse
transcription of 1 mg of RNA was performed using the ImProm II
RT System kit (Promega, Du ¨bendorf, Switzerland). Quantitative
PCR was performed with a 7500 Fast Real-Time PCR System
(Applied Biosystems, Rotkreuz, Switzerland) using the Power
SYBR Green PCR Master Mix (Applied Biosystems) and primer
pairs listed in Table S1. All samples were tested in triplicates.
Amplifications consisted of a denaturation step at 95uC for 15 sec
and an annealing/extension step at 60uC for 60 sec, with the 9600
Emulation mode. For each measurement, a standard made of
successive dilutions of a reference cDNA was processed in parallel.
Gene specific expression was expressed relative to the expression
of HPRT in arbitrary units (A.U.). Gene specific over HPRT ratios
were validated using the house-keeping gene ACTB (human
studies) or Gapdh and Actg1 (mouse studies).
Transfection and reporter assay
THP-1 cells were seeded at 56104cells per well in 24-well plates.
The following day, cells were transiently transfected with 700 ng of
multimeric kB site  and IFNb promoter  luciferase reporter
vectors together with 70 ng of a Renilla luciferase control vector
(Promega) using jetPEITMtransfection reagent (Polyplus-transfec-
tion SA, Illkirch, France). Twenty-four h after transfection, cells
were infected with MVA. Luciferase and Renilla luciferase activities
were measured 24 h latter using the Dual-LuciferaseTM Reporter
Assay System (Promega). Results were expressed as relative
luciferase activity (the ratio of luciferase to Renilla luciferase activity).
Western blot analysis
THP-1 cells were washed with ice cold PBS and lysed for 5 min at
4uC with the M-PER Mammalian Protein Extraction Reagent
(Pierce Biotechnology Inc, Rockford, IL). Reaction mixtures were
centrifuged 5 min at 14’000 rpm. Protein concentration of superna-
tants was determined using the bicinchoninic acid protein assay
(Pierce Biotechnology). Cell-lysates were electrophoresed through
12% (w/v) polyacrylamide gels and transferred onto nitrocellulose
membranes (Schleicher & Schuell, Keene, NH). Membranes were
incubated with antibodies directed against RIG-1, MDA-5, IPS-1
(Apotech Corporation, Epalinges, Switzerland), cleaved IL-1b, total-
and phospho-p44/42 (ERK1/2), and -JNK MAP Kinases, phospho-
IRF3 (Cell Signalling Technology, Danvers, TX), caspase 1 (Santa
Cruz, Santa Cruz, CA), phospho-STAT-1 (BD Biosciences), IRF7
(Zymed, San Franciso, CA) and tubulin (Sigma). After washing,
membranes were incubated with horse radish peroxidase (HRP)-
conjugated secondary antibody (Pierce). Signals were revealed using
the ECL Western blotting Analysis System (GE Healthcare).
Electrophoretic mobility shift assay (EMSA)
Nuclear extracts were prepared and analyzed by EMSA .
Briefly, protein concentration of cell extracts was measured using
the Bradford-dye assay (Bio-Rad). Two mg of nuclear extracts were
incubated for 15 min at room temperature with a radio-labeled
consensus NF-kB probe (Santa Cruz). Reaction mixtures were
electrophoresed through 6% non-denaturing polyacrylamide gels.
Gels were dried and exposed to X-ray films. Supershift
Innate Immune Sensing of MVA
PLoS Pathogens | www.plospathogens.org12 June 2009 | Volume 5 | Issue 6 | e1000480
experiments using anti-p65 antibody (sc-109, Santa Cruz) were
performed as previously described  (data not shown).
Comparisons among treatment groups were performed by two-
tailed paired Student’s t-test. p values less than 0.05 were
considered to indicate statistical significance.
control, TLR2, MDA-5, RIG-I, IPS-1, NALP3 and caspase-1
shRNAs (C, E) were infected with MVA and NYVAC (MOI 5).
Cytokines, chemokines and IFNb production were assessed by
ELISA (A, E). IL-8, IFNb, IP-10, MIP-1a and RANTES mRNA
levels were analyzed by RT-PCR and expressed as reported in
Figure 1 (B, C). Electrophoretic mobility shift assay of NF-kB DNA
(P-STAT-1) and tubulin(D,F).Dataaremeans6SDoftriplicate(A,
B, C, E) samples from one experiment and are representative of two
to three independent experiments. ND: not detected (A).
Found at: doi:10.1371/journal.ppat.1000480.s001 (0.12 MB PDF)
Comparison of macrophage responses to MVA and
IL-8 and IFNb by human macrophages infected with the Western
Reserve strain of vaccinia (WR). THP-1 cells (A–B) and THP-1
cells stably expressing control, TLR2, MDA-5, RIG-I and IPS-1
shRNAs (C) were infected with WR (MOI 5). IL-8 and IFNb
mRNA levels were analyzed by RT-PCR and expressed as
reported in Figure 1 (A and C). IL-8 and IFNb production by
THP-1 cells stimulated for 24 h with WR were assessed by ELISA
(B). Data are means6SD of triplicate samples from one
experiment and are representative of two independent experi-
ments. shTLR2 THP-1 cells produced significantly less IL-8,
whereas shMDA-5 and shIPS-1 THP-1 cells produced signifi-
cantly less IFNb than control cells (C) (p,0.05).
Found at: doi:10.1371/journal.ppat.1000480.s002 (0.02 MB PDF)
TLR2 and MDA-5 contribute to the production of
independent chemokines by THP-1 macrophages infected with
MVA. THP-1 cells stably expressing control and candidate shRNA
(#1 and #2) directed against TLR2 were obtained as described in
Materials and Methods. (A) TLR2 mRNA content was analyzed by
RT-PCR. Results are expressed as the ratio of TLR2 mRNA levels
to that of HPRT. (B) shControl and shTLR2 THP-1 cells were
infected with MVA (MOI 5 unless specified) or stimulated with
Pam3CSK4(1 mg/ml) for 24 h unless otherwise specified. IL-8,
MIP-1a, IFNb and IP-10 mRNA contents were analyzed by RT-
PCR. Results are expressed as the ratio of IL-8, MIP-1a, IFNb and
IP-10 mRNA levels to that of HPRT. (C) IL-8 concentrations were
TLR2 contributes to the production of IFNb-
measured by ELISA. Data are means6SD of triplicate samples
from one experiment and are representative of three (A, B) or two
(C) independent experiments. AU: arbitrary units. shTLR2 THP-1
cells produced significantly less IL-8 and MIP-1a mRNA (B) and
IL-8 protein than control cells (C) (p,0.05).
Found at: doi:10.1371/journal.ppat.1000480.s003 (0.02 MB PDF)
expression in vivo. BALB/c mice were injected i.p. with MVA (107
PFU). Peritoneal cells (A) and splenocytes (B ) were isolated 12 h
after infection as described in Materials and Methods. RIG-I and
MDA-5 mRNA contents were analyzed by RT-PCR. Results are
expressed as the ratio of RIG-I and MDA-5 mRNA levels to that
of HPRT. AU: arbitrary units. Data are means6SD of triplicate
samples from one experiment comprising three mice per
experimental condition and are representative of two independent
experiments (p,0.05 for all conditions).
Found at: doi:10.1371/journal.ppat.1000480.s004 (0.01 MB PDF)
MVA infection increases RIG-I and MDA-5 mRNA
of RIG-I, MDA-5 and IPS-1. THP-1 cells stably expressing
control and candidate shRNA (#1 and #2) directed against RIG-
I, MDA-5 and IPS-1 were obtained as described in Materials and
Methods. RIGI, MDA-5 and IPS-1 mRNA contents were analyzed
by RT-PCR and expressed as reported in Figure 1. Data are
means6SD of triplicate samples from one experiment and are
representative of two independent experiments.
Found at: doi:10.1371/journal.ppat.1000480.s005 (0.01 MB PDF)
Generation of THP-1 cells expressing reduced levels
of the IRF3 transcription factor and IFNb secretion. THP-1 cells
stably expressing control, NALP3, ASC and caspase 1 (casp1)
shRNAs were infected with MVA (MOI 5 unless specified otherwise)
(A, B) or cultured with (+) or without (2) monosodium urate
monohydrate (MSU) cristals crystals for the indicated time (A) or 6 h
(B, C). Western blots of intracellular phosphorylated IRF3 and
tubulin (A) and IFNb (B) and IL-1b (C) concentrations measured by
ELISA in cell-culture supernatants. Data are means6SD of triplicate
samples from one experiment and are representative of two
independent experiments. p,0.05 for cells transduced with NALP3,
ASC and casp1 shRNAs vs. control shRNA (C).
Found at: doi:10.1371/journal.ppat.1000480.s006 (0.12 MB PDF)
The NALP3 inflammasome is dispensable for activation
Found at: doi:10.1371/journal.ppat.1000480.s007 (0.02 MB PDF)
Oligonucleotides used in RT-PCR analyses.
Conceived and designed the experiments: JD TR QGST TC. Performed
the experiments: JD DLR MKR. Analyzed the data: JD TR QGST DLR
TC. Contributed reagents/materials/analysis tools: JD TR QGST SA VP
CEG BP JT GP ME TC. Wrote the paper: JD TR ME TC.
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