Hepatitis C Virus Fails To Activate NF-?B Signaling in Plasmacytoid
Clélia Dental,a,b,cJonathan Florentin,a,b,cBesma Aouar,a,b,cFrancoise Gondois-Rey,a,b,cDavid Durantel,dThomas F. Baumert,e
Jacques A. Nunes,a,b,cDaniel Olive,a,b,cIvan Hirsch,a,b,cand Ruzena Stranskaa,b,c
Institut National de la Santé et de la Recherche Médicale (Inserm) UMR891, Centre de Recherche en Cancérologie de Marseille, Marseille, Francea; Institut Paoli-Calmettes,
Marseille, Franceb; Université de la Méditerranée, Aix-Marseille II, Marseille, Francec; Inserm Unit 1052, CNRS UMR5286, Centre de Recherche en Cancérologie de Lyon
(CRCL), Lyon, Franced; and Inserm U748, Université de Strasbourg, Institute de Virologie, Strasbourg, Francee
infection and are responsible for production of type I interferons
ing viral infection (15, 19, 32). pDCs are able to detect the genetic
material of viruses with a subset of Toll-like receptors (TLR) lo-
calized to the endosomal compartment (10). These nucleotide-
sensing TLRs include TLR7 and TLR8, which recognize single-
stranded RNA, and TLR9, which recognizes DNA. TLR7 also
recognizes synthetic imidazoquinoline components, for example
R848 (resiquimod), whereas TLR9 recognizes synthetic CpG
and TLR9 with their agonists triggers a signaling cascade, which
starts with recruitment of the MyD88 adaptor molecule to the
assembly of a multiprotein signal-transducing complex in the cy-
toplasm that includes interferon-regulatory factor 7 (IRF7) (10).
Activated IRF7, which is constitutively expressed in pDCs, trans-
The elimination of hepatitis C virus (HCV) in more than 50%
(IFN-?) (9, 20) suggests that pDCs can play an important role in
the control of HCV infection. Several reports have shown that
no or only weak production of type I IFN and cell differentiation
(4, 7, 11, 13, 31). A recent report has shown that pDCs exposed in
direct cell-to-cell contact with HCV-infected hepatoma cells, un-
like those exposed to cell-free HCV virions, produce large
amounts of type I IFN via TLR7 signaling (35). This suggests that
pDCs could be responsible for production of intrahepatic type I
lasmacytoid dendritic cells (pDCs) are a highly specialized
subset of dendritic cells that function as sentinels for viral
IRF7-mediated production of IFN-?, MyD88 signaling also leads
to activation of nuclear factor kappa B (NF-?B) and mitogen-
activated protein kinases (MAPKs). Both NF-?B and MAPKs
stimulate secretion of the proinflammatory cytokines interleukin
6 (IL-6) and tumor necrosis factor ? (TNF-?) and stimulate ex-
pression of costimulatory molecules such as CD80 and CD86.
TLR7 and dependent on PI3K-p38MAPK, which stimulates the
apoptosis-inducing ligand (TRAIL) in the absence of type I IFN
To better understand the molecular mechanism of HCV sens-
ing, we investigated whether exposure of pDCs to HCV-infected
hepatoma cells induces not only IRF7 signaling but also NF-?B
that in comparison to influenza virus or synthetic agonists of
TLR7 and TLR9, HCV-infected hepatoma cells did not stimulate
in pDCs phosphorylation of NF-?B and activation of NF-?B-
dependent pDC responses, such as cell surface expression of dif-
Received 17 June 2011 Accepted 1 November 2011
Published ahead of print 16 November 2011
Address correspondence to Ruzena Stranska, firstname.lastname@example.org, or Ivan
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jvi.asm.org0022-538X/12/$12.00 Journal of Virologyp. 1090–1096
ferentiation markers CD40, CCR7, CD86, and TRAIL and secre-
tion of TNF-? and IL-6. In contrast, production of TNF-? and
IL-6 in pDCs exposed to the HCV-infected hepatoma cells was
induced by CpG-A and CpG-B, showing that HCV-infected hep-
atoma cells did not actively inhibit TLR-mediated NF-?B phos-
phorylation. Our results suggest that cell-associated HCV signals
in pDCs via an endocytosis-dependent mechanism and IRF7 and
results are thus important for an understanding of the HCV-DC
chronic HCV infection.
MATERIALS AND METHODS
Isolation and culture of pDCs. Peripheral blood mononuclear cells
(PBMCs) from healthy anonymous donors were obtained from the na-
tional blood services (Etablissement Francais du Sang [EFS], Marseille,
France). pDCs purified from PBMCs as described previously (4, 11, 13,
than 5% myeloid dendritic cells. Isolated pDCs were cultured in RPMI
viability, recombinant IL-3 (R&D Systems Europe, Ltd., Abingdon,
United Kingdom) was added to a final concentration of 10 ng/ml.
M). Cell culture-derived HCV (HCVcc) virions were prepared in Huh7.5
cells (2) (kindly provided by APATH L.L.C.) on the basis of plasmid
pJFH-1 displaying mutations F172C and P173S in core, and N534K in E2
(5), as described previously (4, 11, 13, 31). The ultracentrifuged virus
medium to obtain a 1,000-fold-concentrated virus suspension.
containing virus particles were determined routinely with quantitative
reverse transcription-PCR (RT-PCR) as described previously (4, 11, 13,
31) using the primer RTU1 (25) for cDNA synthesis, primer pair UTR2
and RTU2 (25) for PCR amplification, and HCV JFH-1 RNA prepared in
vitro with T7 polymerase as a standard.
Influenza virus stocks. Influenza virus A/H3N2/Johannesburg/34/99
was produced in MDCK cells (12).
Huh7.5 infected with JFH-1 3 M at a multiplicity of infection of 0.01
focus-forming units (FFU)/cell were cultured under standard conditions
for 1 week. The percentage of infected cells was determined by immuno-
fluorescence staining for HCV core protein using a mouse monoclonal
anti-core antibody (C7-50) (Thermofisher Scientific, Courtaboeuf,
France). Monolayers containing ?60% HCV core?Huh7.5 cells were
used in coculture with pDCs. Huh7.5 cells transfected with H/SG-neo
(L?I) subgenomic replicon (SGR) (2) (kindly provided by C. M. Rice,
The Rockefeller University, New York, NY) and virus-free Huh7.5 cells
were used as controls.
of 106cells/ml aliquoted in 100-?l quantities in 96-well round-bottom
(ODN 2006), or CpG-C (ODN 2395) (InvivoGen, San Diego, CA), 5 ?M
R848 (InvivoGen, San Diego, CA), 10 ng/ml TNF-? (Becton Dickinson,
Le Pont-De-Claix, France), HCV JFH-1 3 M virions at a multiplicity of
100 virus genome copies per cell, or Huh7.5 cells infected with HCV
JFH-1 3 M or transfected with SGR at the ratio of 2 Huh7.5 cell per pDC,
for 20 or 40 h in the presence of IL-3. Endocytosis and endosomal matu-
ration/acidification was inhibited by chlorpromazine (6.25 ?g/ml) or
chloroquine (5 ?M), both from Sigma-Aldrich.
pression, cells were incubated for 15 min at room temperature in the
presence of V450-conjugated CD86, fluorescein isothiocyanate (FITC)-
conjugated Lineage Cocktail 1 (CD3, CD14, CD16, CD19, CD20, CD56),
PercP-Cy5.5-conjugated CD123, PE-Cy7-conjugated CCR7, APC-
conjugated CD11c and APC-H7-conjugated CD40 monoclonal antibod-
ies (MAbs) (all Becton Dickinson), and biotin-conjugated TRAIL MAb
(eBioscience, Paris, France) with second-step reagent phycoerythrin
(Becton Dickinson). After labeling, cells were fixed with 4% paraformal-
dehyde and analyzed using the LSR II instrument (Becton-Dickinson, Le
were analyzed with Flowjo software (Tree Star, Inc., Ashland, OR).
Determination of secreted IFN-?, TNF-?, and IL-6. The quantities
of total IFN-?, TNF-?, and IL-6 produced by pDCs were measured in
(ELISA) kits (IFN-? from eBiosciences, Paris, France, and TFN-? and
IL-6 from Becton-Dickinson, Le Pont de Claix, France, respectively).
Determination of NF-?B p65 phosphorylation by dynamic
phospho-flow cytometry. Phospho-flow analysis of phosphorylation of
NF-?B p65 at serine 536 was performed as previously described (8).
Briefly, cells were fixed, permeabilized, and incubated successively with
Phospho-NF-?B p65 (Ser536) (93H1) rabbit MAb (Cell Signaling, Dan-
vers, MA) and anti-rabbit biotinylated antibodies. Finally, detection was
Statistical analysis. Quantitative variables are expressed as the
means ? standard errors of the means (SEM). To compare the levels of
metric Mann-Whitney test with GraphPad Prism 4 software (GraphPad
Software, La Jolla, CA). All tests of significance were two-sided, and a P
value of ?0.05 was considered to be significant.
production of IFN-? but not of TNF-?. First, we compared the
subgenomic replicon (HCV SGR) with the levels of IFN-? in-
duced by HCV JFH-1 virions, influenza virus A/H3N2/Johannes-
burg (Flu), R848, CpG-A, CpG-B, and CpG-C. Stimulation of
pDCs with Huh7.5 cells infected with HCV JFH-1 or transfected
with SGR or stimulation with the TLR7 agonists R848 and Flu,
CpG-C strongly increased production of IFN-? (Fig. 1). In con-
trast, stimulation of pDC with HCV JFH-1 virions or CpG-B did
not increase the production of IFN-? in comparison with non-
stimulated or control Huh7.5-stimulated pDCs. Thus, consistent
with previous results (35), cell-associated HCV does but cell-free
HCV does not induce production of IFN-? in pDCs. In addition
to IFN-?, we also investigated the release of the proinflammatory
cytokine TNF-? in the same experimental system. Production of
TNF-? was strongly stimulated with CpG-A, CpG-B, CpG-C,
R848, and Flu. In contrast to results for IFN-?, production of
(P ? 0.0004) or transfected with HCV SGR (P ? 0.0018) was
Exposure to hepatoma cells infected with HCV does not in-
duce phosphorylation of NF-?B p65 in pDCs. The absence of
TNF-? in cell-free supernatants of pDCs exposed to cell-
which is known to control production of proinflammatory cyto-
kines and differentiation of pDCs (10). First, we compared the
Huh7.5 cells infected with HCV JFH-1 or transfected with HCV-
SGR with the levels induced by R848, Flu, CpG-B, or HCV JFH-1
NF-?B Signaling in HCV-Exposed pDCs
January 2012 Volume 86 Number 2jvi.asm.org 1091
virions (Fig. 2). Stimulation of pDCs with R848, Flu, or CpG-B
strongly increased phosphorylation of NF-?B p65. In contrast,
stimulation of pDC with HCV JFH-1 virions or with Huh7.5 cells
infected with HCV JFH-1 or transfected with HCV SGR revealed
no increase of phosphorylated NF-?B p65 in comparison with
control nonstimulated or Huh7.5-stimulated pDCs (Fig. 2A and
B). These findings show that neither cell-free nor cell-associated
HCV induced phosphorylation of NF-?B p65 in pDCs.
Exposure to hepatoma cells infected with HCV does not in-
associated with the pDC cross-presentation function. To address
this question, we compared the impact on pDC differentiation
induced by Huh7.5 cells infected with HCV JFH-1 or transfected
CD40, CD86, CCR7, and TRAIL in CD11c?CD123?gated live
cells (Fig. 3A). Culture of pDCs in medium supplemented with
donors stimulated with TLR7 agonist R848 or TLR9 agonist
CpG-B showed strongly increased expression of CD40, CD86,
Huh7.5 cells infected with HCV JFH-1 or transfected with HCV
SGR did not result in an increase of expression of CD40, CD86,
CCR7, and TRAIL; the same pDC phenotype was observed with
HCV JFH-1 virions.
Additional analysis of the kinetics of expression of differentia-
tion markers of pDCs stimulated with R848 and CpG-B con-
firmed significant upregulation of CD40 (PR848 ? 0.0022,
PCpG-B? 0.0048; P values for pDCs stimulated with R848 and
PCpG-B? 0.0048), and TRAIL (PR848? 0.0046, PCpG-B? 0.0046)
negative controls was observed in pDCs exposed to HCV virions
SGR. Taken together, these findings show that neither cell-free
HCV nor cell-associated HCV induces differentiation of pDCs.
Exposure of pDCs to HCV-infected hepatoma cells does not
impair TLR-dependent or independent phosphorylation of
NF-?B and subsequent production of TNF-? and IL-6. Finally,
we investigated whether HCV-infected hepatoma cells elicit in
pDCs a dominant negative effect on the phosphorylation of
NF-?B and subsequent NF-?B-dependent pDC responses or are
just weak inducers of the NF-?B signaling pathway (Fig. 4). To
address this question, we stimulated pDCs with CpG-B (via a
FIG 1 IFN-? and TNF-? production by coculture of pDCs with HCV-
infected hepatoma cells. pDCs were cocultured with HCV-infected Huh7.5
cells (Huh-HCV), with Huh7.5 cells transfected with HCV subgenomic repli-
cons (Huh-SGR) or with control Huh7.5 cells (Huh), were inoculated with
were stimulated with CpG-A, CpG-B, CpG-C, or R848. Secretion of IFN-?
(light columns) and TNF-? (dark columns) in cell-free supernatant was de-
termined by ELISA 20 h poststimulation. The data show means and SEM of
results of at least seven independent experiments with pDCs from different
hepatoma cells. (A) pDCs were cocultivated with Huh7.5 cells infected with
SGR), or with control Huh 7.5 cells (Huh). In parallel, pDCs were inoculated
with HCV JFH-1 virions (HCV-virion) or with influenza virus A/H3N2/Jo-
hannesburg (Flu) and incubated for 60 min. In control experiments, pDCs
determined by phospho-flow cytometry. Shaded areas show nonstimulated
pDC; black lines show stimulated pDCs. Data are representative of results of
parable results. (B) The relative mean fluorescence intensity (MFI) corre-
results of six independent experiments with pDCs from different donors.
Dental et al.
jvi.asm.orgJournal of Virology
TLR9-dependent pathway) or with TNF-? (via a TLR9-
independent pathway), and we investigated the effects of HCV-
infected hepatoma cells on NF-?B phosphorylation. Results of
these experiments show that HCV-infected hepatoma cells
blocked neither CpG-B- nor TNF-?-mediated phosphorylation
of NF-?B (Fig. 4A).
Furthermore, HCV-infected hepatoma cells did not impair
the priming with the cell-free virus precedes stimulation with
7, 11, 13, 31). Therefore, we investigated the effect of priming of
pDCs with HCV-infected Huh7.5 cells followed 2 h later by stim-
ulation with CpG-A or CpG-B on production of TNF-? or IL-6
(Fig. 4C). Even under these experimental conditions, cell-
FIG 3 Expression of differentiation markers of pDCs exposed to HCV-infected hepatoma cells. (A) Gating strategy for identification of pDCs: magnetic
of Lin1?CD11c?cells into a CD123?pDC population. The purity of the isolated pDCs was determined from flow cytometry analysis as a fraction of Lin1?,
CD123?, and CD11c?cells. Numbers displayed in delimited areas are percentages of positive cells. (B and C) The expression of the cell surface markers CD40,
CD86, and CCR7 was determined immediately after cell separation (0 h, shaded area), after 20 h culture (gray line), or after 40 h culture (black line). (B) pDC
differentiation in the presence of IL-3. (C) pDCs were cocultured with Huh7.5 cells infected with HCV JFH-1 (Huh-HCV) or transfected with HCV SGR
(Huh-SGR), were cocultured with control Huh7.5 cells (Huh), or were inoculated with cell-free HCV JFH-1 (HCV-virion). In control experiments, pDCs were
results. (D) Kinetics of expression of differentiation markers of pDCs exposed to HCV. Percentages of stimulated CD40?, CCR7?, CD86?, and TRAIL?cells
relative to nonstimulated cells at the time zero are shown. The data show means and SEM of six (20 h) and three (40 h) independent experiments with pDCs of
different donors stimulated for 0, 20 (n ? 6), and 40 (n ? 3) h. ??, 0.05 ? P ? 0.01.
NF-?B Signaling in HCV-Exposed pDCs
January 2012 Volume 86 Number 2 jvi.asm.org 1093
FIG4 Effect of HCV-infected hepatoma cells on TLR-dependent and TLR-independent phosphorylation of NF-?B and on NF-?B-dependent functions.
pDCs were stimulated with CpG-A, CpG-B, or TNF-? in the presence of HCV JFH-1-infected Huh7.5 cells (Huh-HCV) or control Huh7.5 cells (Huh)
or in the presence of endocytosis inhibitors. (A) Phosphorylation of NF-?B p65 was determined by phospho-flow cytometry. The relative MFI corre-
sponds to the MFI of cells stimulated for 30 min divided by the respective MFI values of nonstimulated cells. The data show means and SEM of results of
five independent experiments with pDCs from different donors. (B and C) Levels of TNF-?, IL-6, and IFN-? in the cell-free supernatants of pDCs from
at least six donors were determined by ELISA, either after simultaneous exposure of pDCs to Huh-HCV or Huh stimulated with CpG-A or CpG-B (B) or
after priming with Huh-HCV or Huh that was followed 2 h later by stimulation with CpG-A or CpG-B (C). In a similar way production of IFN-? was
determined in pDCs exposed to Huh-HCV or CpG-A that were simultaneously (D) or 2 h later (E) treated with inhibitors of endocytosis, chlorpromazine
(CHP), or chloroquine (CHQ). For a better comparison of the effect of endocytosis inhibitors, IFN-? production was expressed as a percentage of the
production triggered by the control agonists without inhibitors.
Dental et al.
jvi.asm.orgJournal of Virology
associated HCV did not block production of proinflammatory
in contrast to HCV particles (4, 7, 11, 13, 31), HCV-infected
Huh7.5 cells did not inhibit production of IFN-? by pDCs stim-
to activate NF-?B signaling in pDCs without having a dominant
negative effect on NF-?B phosphorylation induced by other acti-
Endocytosis is relevant for sensing of HCV-infected hepa-
of cell-associated HCV by pDCs, we investigated the functional
impact of endocytosis on virus recognition. To address this ques-
tion, we tested several inhibitors impairing cellular endocytosis
pathways. Inhibitors were mixed with pDCs, either simultane-
the priming (Fig. 4E). Chlorpromazine (CHP), an inhibitor of
clathrin-coated pit-mediated endocytosis, inhibited production
of IFN-? to 6.3% when pDCs were simultaneously exposed to
HCV-infected Huh7.5 cells and endocytosis inhibitor, while pro-
of pDCs with chloroquine (CHQ), a lysosomotropic weak base
that neutralizes the acidic environment of endocytic vesicles, re-
uration of the endosomes is necessary for activation of pDCs. In-
hibitors of endocytosis used in these experiments did not change
the background levels of proinflammatory cytokines TNF-? and
IL-6 induced by exposure of pDCs to HCV-infected Huh7.5 cells,
although they efficiently blocked production of proinflammatory
cytokines induced by CpG-A (not shown). In conclusion, our
findings suggest that endocytosis of viral or cellular structures of
somes play a role in pDC activation.
Our results demonstrate that HCV-infected hepatoma cells trig-
ger the IRF7 signaling pathway but not signaling via NF-?B in
pDCs. Despite robust production of IFN-? by pDCs exposed to
HCV-infected cells, these cells did not induce significant phos-
phorylation of the p65 subunit of NF-?B or production of the
proinflammatory cytokines TNF-? and IL-6. Moreover, HCV-
infected cells did not induce a significant expression of differenti-
ation markers CD40, CCR7, CD86, or TRAIL. These results sug-
gest that cell-associated HCV does not stimulate a full functional
HIV where CD4?T cell-associated virus was shown to induce a
strong IFN-? response in pDCs but only a weak expression of
pDC activation and differentiation markers (18, 30).
for effective cross-presentation by pDCs (22). TRAIL plays an
essential role in their cytotoxicity (3). These functions facilitate
the central role of pDCs in linking innate and adaptive immune
responses. Our results are consistent with observations that sug-
gest that this link might be impaired in HCV infection (1, 37, 38).
These ex vivo studies performed with patients with chronic HCV
infections reported decreased numbers and impaired function of
virus-specific CD4 and CD8 T cells, suggesting a defect in pDC
function. Low plasma levels of IFN-? and of proinflammatory
cytokines, together with a delayed systemic immune response,
were also observed in acute HCV infection, in comparison with
levels in human immunodeficiency virus 1 (HIV-1) infection
(33). In addition, studies investigating responses of pDCs from
healthy donors concluded that HCV does not induce production
of proinflammatory cytokines and cell differentiation (4, 11, 31).
Exposure of pDCs from healthy donors to cell-free HCV par-
ticles (4, 7, 11, 13, 31) or cell-associated virus did not induce cell
differentiation and production of proinflammatory cytokines.
The major difference between both viral systems lies in the pro-
duction of type I IFN, induced in pDCs exclusively by the cell-
associated virus (35), and the blockade of TLR9-mediated pro-
We hypothesize that the sensing of the cell-associated virus via
TLR7 (35), in contrast to the sensing of the HCV particles by
regulatory receptors of pDCs, is responsible for this difference
(13). A high level of variability of analyzed markers among differ-
ent donors in both viral systems could reflect genetic polymor-
phism of the determinants of response to HCV infection. This
variability could result in different outcomes of HCV infection
variables, such as sex (21), age (24, 34), and race (36) that were
previously shown to be important for pDC function remains un-
disclosed in our present study with anonymous healthy donors.
signaling pathway in pDCs is compatible with the mechanism of
spatiotemporal regulation of TLR signaling. This mechanism is
based on the different compartmentalization and retention time
of TLR ligands within the endolysosomal pathway (14, 16, 39).
According to this concept, TLR signaling in early endosomes (in-
duced for example by A-type and C-type CpG DNA) is coupled
with production of type I IFN, whereas signaling from late endo-
somes (induced for example by B-type and C-type CpG DNA) is
tory cytokines (14). As expected, our findings show that endocy-
tosis and subsequent acidification of endosomes are relevant for
pDC activation by HCV-infected hepatoma cells.
The concept of spatiotemporal regulation was recently modi-
selective receptor trafficking within the endosomal system (29).
Whereas Flu and R848 signal, according to this model, from both
early (NF-?B and IRF7) and late endosomes and stimulate pro-
only via IRF7 endosomes to stimulate production of type I IFN.
Preincubation of pDCs with HCV-infected hepatoma cells prior
to TLR9 stimulation did not alter the production of IFN-? and
proinflammatory cytokines. This contrasts with the inhibitory ef-
fect of cell-free HCV on IFN-? production (11) and suggests dif-
ferent spatiotemporal regulation of TLR signaling employed by
the two forms of virus (13). A recent report has shown that HIV
particles preferentially traffic to the early IRF7 endosomes in
pDCs and therefore skew pDCs toward a partially matured, per-
sistently IFN-?-secreting phenotype (23).
In infected liver, pDCs are exposed to both hepatocyte-associated
HCV and cell-free HCV virions. pDCs exposed to hepatocyte-
associated HCV are good candidates for production of intrahe-
NF-?B Signaling in HCV-Exposed pDCs
January 2012 Volume 86 Number 2jvi.asm.org 1095
interferon-stimulated genes (ISGs) but also promotes greater ac-
tivation of both pDCs and myeloid dendritic cells during virus
infections (26). Production of type I IFN and efficient antigen
processing and presentation for specific stimulation of T cells are
both necessary for clearing HCV infection. Thus, incomplete ac-
tivation of pDCs during chronic hepatitis C might be a reason for
their inappropriate responsiveness. We found that the HCV-
infected hepatoma cells did not actively block NF-?B phosphory-
lation and that full response of pDCs exposed to cell-associated
virus could be induced by nucleotide-sensing TLR agonists. In
addition to TLR agonists, NF-?B phosphorylation in pDCs ex-
posed to cell-associated virus can be induced also via a TLR-
independent pathway, e.g., by TNF-?. The finding that CpG-A
and CpG-B can restore NF-?B-dependent functions that are not
induced in pDCs exposed to cell-associated HCV suggests a po-
tential perspective of TLR agonists to improve response to antivi-
ral therapy. In view of the central role of pDCs in regulating the
ment of chronic HCV infection.
Our work was supported by grants from the French National Agency for
la Recherche Médicale), Institut Paoli Calmettes, and Plateform Cancer
decision to publish, or preparation of the manuscript.
We thank T. Wakita for providing the HCV JFH-1 clone and C. M.
Rice for the H/SG-neo (L?I) subgenomic replicon.
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