of June 13, 2013.
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Plasmacytoid Dendritic Cell Loss in Chronic
Mechanisms of Reduced IFN-
Hepatitis C Virus (HCV) Core
GyongyiMandrekar, Gennadiy Bakis, Maureen Cormier and
Angela Dolganiuc, Serena Chang, Karen Kodys, Pranoti
2006; 177:6758-6768; ;
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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Immunologists All rights reserved.
Copyright © 2006 by The American Association of
9650 Rockville Pike, Bethesda, MD 20814-3994.
The American Association of Immunologists, Inc.,
is published twice each month by
The Journal of Immunology
by guest on June 13, 2013
Hepatitis C Virus (HCV) Core Protein-Induced,
Monocyte-Mediated Mechanisms of Reduced IFN-? and
Plasmacytoid Dendritic Cell Loss in Chronic HCV Infection1
Angela Dolganiuc, Serena Chang, Karen Kodys, Pranoti Mandrekar, Gennadiy Bakis,
Maureen Cormier, and Gyongyi Szabo2
IFN-? production by plasmacytoid dendritic cells (PDCs) is critical in antiviral immunity. In the present study, we evaluated the
IFN-?-producing capacity of PDCs of patients with chronic hepatitis C virus (HCV) infection in treatment-naive, sustained
responder, and nonresponder patients. IFN-? production was tested in PBMCs or isolated PDCs after TLR9 stimulation. Treat-
ment-naive patients with chronic HCV infection had reduced frequency of circulating PDCs due to increased apoptosis and
showed diminished IFN-? production after stimulation with TLR9 ligands. These PDC defects correlated with the presence of
HCV and were in contrast with normal PDC functions of sustained responders. HCV core protein, which was detectable in the
plasma of infected patients, reduced TLR9-triggered IFN-? and increased TNF-? and IL-10 production in PBMCs but not in
isolated PDCs, suggesting HCV core induced PDC defects. Indeed, addition of rTNF-? and IL-10 induced apoptosis and inhibited
IFN-? production in PDCs. Neutralization of TNF-? and/or IL-10 prevented HCV core-induced inhibition of IFN-? production.
We identified CD14?monocytes as the source of TNF-? and IL-10 in the HCV core-induced inhibition of PDC IFN-? production.
Anti-TLR2-, not anti-TLR4-, blocking Ab prevented the HCV core-induced inhibition of IFN-? production. In conclusion, our
results suggest that HCV interferes with antiviral immunity through TLR2-mediated monocyte activation triggered by the HCV
core protein to induce cytokines that in turn lead to PDC apoptosis and inhibit IFN-? production. These mechanisms are likely
to contribute to HCV viral escape from immune responses. The Journal of Immunology, 2006, 177: 6758–6768.
with ribavirin is currently the most effective treatment for patients
with hepatitis C infection (1, 2). The broad repertoire of the bio-
logical effects of IFN-? include, but are not limited to, direct in-
hibition of viral replication in infected cells, enhancement of cy-
totoxic activity of macrophages and NK cells, promotion of the
survival of T cells, and Ab production by B cells (3). Although
much controversy exists regarding the dose, duration, and effec-
tiveness of IFN therapy, it can lead to viral elimination and reso-
lution of systemic inflammation (1, 2, 4). In vivo, the main pro-
ducers of IFN-? are plasmacytoid dendritic cells (PDCs). Human
PDCs are lineage (CD3, CD8, CD11c, CD14, CD19, and CD56)-
negative cells that express CD4, CD123, and BDCA-2 markers on
their surface and produce high levels of type I IFNs (IFN-??)
when exposed to viruses or stimulated with certain TLR ligands.
PDCs express TLR1, TLR6, TLR7, and TLR9, of which TLR9 is
the most prominent (5–7). Current reports about the IFN-?-pro-
nfection with hepatitis C virus (HCV)3becomes chronic in
most cases (50–80%) and causes liver damage ranging from
mild inflammation to cirrhosis (1). IFN-? in combination
ducing capacity of HCV-infected patients are controversial. Both
reduced and increased IFN-? production was reported in HCV-
infected patients (8–11).
The aim of the present study was to analyze PDC functions in
patients with chronic HCV infection. Our results indicate a loss of
circulating PDCs due to apoptosis associated with reduced IFN-?
production in HCV-infected patients. We identified that HCV core
protein can impair IFN-? production by PDCs via monocyte-de-
rived IL-10 and TNF-? induction. Our findings reveal a novel
HCV-driven mechanism for PDC loss that may provide an escape
mechanism for HCV from immune surveillance.
Materials and Methods
Blood donors and cells
Healthy individuals (controls, n ? 46), treatment-naive patients with
chronic infection (HCV patients, n ? 41), patients who cleared HCV (sus-
tained responders, n ? 16), and those who failed to clear HCV (nonre-
sponders, n ? 14) after therapy were enrolled in the study (Table I). Of
responders, 12 were treated with Pegasus plus ribavirin for 24–48 mo, 1
received Pegasus plus ribavirin plus viramidin for 36 mo, 2 had polyeth-
ylene glycol-intron plus ribavirin for 48 mo, and 1 was treated with infer-
gen15 plus ribavirin for 62 mo. The responders were enrolled in the study
9 mo to 4 years after the last dose of therapeutic agent and all have been
HCV-free since. Nonresponders were previously on similar treatment reg-
imens and did not clear the virus upon completion of the treatment; they
were enrolled in the study at least 6 mo after the therapy was completed
and had detectable viral counts upon recruitment into the study. A cohort
of HCV-naive patients with nonalcoholic liver disease (nonalcoholic ste-
atohepatitis (NASH)) with features of liver inflammation (elevated liver
enzymes (alanine aminotransferase), lack of viral infection, and liver bi-
opsy-proven inflammation) was recruited (n ? 6). The study was approved
by the Committee for the Protection of Human Subjects in Research at the
University of Massachusetts, and informed consent was obtained.
PBMCs were separated from peripheral blood using centrifugation in
Ficoll-Paque Plus gradient (Amersham Biosciences), as we described
previously (12). The cells were cultured at 1 ? 106/ml in 96-well plates
University of Massachusetts Medical School, Department of Medicine, Worcester,
Received for publication February 8, 2006. Accepted for publication August 30, 2006.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This research was supported by National Institutes of Health Grant R01 AA14372
2Address correspondence and reprint requests to Dr. Gyongyi Szabo, University of
Massachusetts Medical School, Department of Medicine, LRB 215, 364 Plantation
Street, Worcester, MA 01605. E-mail address: email@example.com
3Abbreviations used in this paper: HCV, hepatitis C virus; PDC, plasmacytoid den-
dritic cell; NASH, nonalcoholic steatohepatitis; PI, propidium iodide; Ct, comparative
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc.0022-1767/06/$02.00
by guest on June 13, 2013
in RPMI 1640 medium (Invitrogen Life Technologies) with 10% FBS
ODN2216 (CpG-A) was from InvivoGen; herpes simplex virus (HSV
KOS-1 strand, UV-irradiated) was a gift from Dr. R. Welsh at the Uni-
versity of Massachusetts (Worcester, MA); rIL-10 and TNF-? were
both from PeproTech. ELISA kits (IL-10 and TNF-?) were from BD
Bioscience and IFN-? from BioSource International. HCV core protein
was purchased from BioDesign. rHCV core protein and core protein
content in the plasma of patients were quantified using the Ortho HCV
core Ag ELISA test kit (Wako Chemicals). The concentrations of the
rHCV core protein are indicated based on the results determined in the
HCV core ELISA. The rHCV core protein preparation was stabilized
with ?-galactosidase; thus, ?-galactosidase, expressed and purified
identically to HCV protein (BioDesign), was used as a negative control.
LPS contamination of recombinant proteins was ?0.01 EU/ml (by
Limulus amebocyte assay; BioWhittaker). Blocking anti-TNF-?, iso-
type controls, and anti-TLR2 and anti-TLR4 Abs were purchased from
eBioscience; anti-IL-10 Ab was from BioSource International.
PDCs were isolated from PBMCs using anti-BDCA2 Abs, according
to the manufacturer’s recommendations (Miltenyi Biotec). Briefly,
PBMCs (108cells/100 ?l of separation buffer (ice-cold PBS, 0.5% BSA,
and 2 mM EDTA)) were incubated with FcR-blocking reagent (50 ?l/108
cells) and anti-BDCA-2-biotin Ab (100 ?l/108cells) for 10 min at ?4°C,
followed by addition of 400 ?l of separation buffer/108cells, FcR-blocking
reagent (150 ?l/108cells) and anti-biotin MicroBeads (200 ?l/108cells) for
15 min at ?4°C. After incubation, PBMCs were washed with 20 volumes
of cold separation buffer and filtered through a magnetic column. To in-
crease the purity of BDCA2?PDCs, the magnetic separation was repeated
using a fresh column. The purity of the isolated PDCs, determined by flow
cytometry analysis after staining with streptavidin-PE and anti-CD123-
allophycocyanin Abs, was ?95% (data not shown). For IFN-? production
and apoptosis studies, PDCs were cultivated for 48 h in RPMI 1640 me-
dium with 10% FBS and 5 ng/ml rIL-3 (PeproTech) in the absence or
presence of TLR9 ligands.
CD14?cells were separated from PBMCs using anti-CD14 Abs-coated
magnetic beads (Mitenyi Biotec), as manufacturer recommended. Briefly,
107PBMCs/80 ?l were incubated with 20 ?l of anti-CD14 Abs-coated
microbeads for 15 min at ?4°C, then washed with 20 volumes of separa-
tion buffer and purified as described for PDCs.
T lymphocytes were purified using the “T cell-negative isolation kit”
(Dynal Biotech), according to manufacturer instructions. Briefly, PBMCs
(1 ? 107/200 ?l of 10% FBS-PBS) were incubated with 20 ?l of Ab mix
(containing anti-CD14, CD16, CD56, HLA-DR/DP, and CD235a) for 10
min at 4°C, washed, and incubated with 100 ?l of depletion Dynabeads for
amino acids (Sigma-Aldrich).
15 min at room temperature. Non-T cells were separated in magnetic field,
whereas T cells were washed and used for coculture with PDCs.
Flow cytometry analysis
For phenotyping, PBMCs were stained with a labeled anti-lineage Abs
(CD3, CD8, CD11c, CD14, CD20, and CD56) and anti-CD4, or anti-
CD123 (all from BD Biosciences) and anti-BDCA-2 (Miltenyi Biotec), or
matching isotype control Abs (BD Biosciences and Miltenyi Biotec), fixed
with 2% paraformaldehyde in PBS, and analyzed by flow cytometry. One
hundred thousand events from the gate corresponding to mononuclear cells
were collected using a FACSCalibur flow cytometer and analyzed with
CellQuest software (BD Biosciences).
PDC apoptosis was detected using the ApoTarget kit (BioSource Inter-
national) and analyzed by flow cytometry. PDCs were gated based on size
and granularity and analyzed for the presence of fluorescent cells.
RNA isolation and real-time PCR
Total RNA was isolated from frozen liver tissue using the RNeasy Micro-
Kit from Qiagen, according to the manufacturer’s instructions. Reverse
transcriptions were performed using the First-Strand cDNA Synthesis kit
(Promega), according to manufacturer’s instructions. One microgram of
total RNA was transcribed to cDNA in 20-?l reaction volume. For tran-
script quantification purposes by real-time PCR, the SYBR Green Mix
containing Thermo-Start DNA Polymerase was used according to the man-
ufacturer’s instructions (Eurogentec). Primers for CD123 (forward 5?-CGA
ACC TAA GGA TGA AAG CAA AGG-3? and reverse 5?-TCG GAC
GGT GTA GTT GGT CAC TTC-3?) and BDCA-2 (forward 5?-TTG AAA
GAA CCA CAC CCC GAA AGT-3? and reverse 5?-TAG CTT TCT ACA
ACG GTG GAT GCC-3?) were from IDT. The 18S primers were pur-
chased from Ambion. The PCR using 1 ?l of cDNA was conducted in
iCycler Thermal Cycler (Bio-Rad). A hot-start phase was applied at 95°C
for 10 min for all primers. cDNA was amplified (45 cycles for CD123 and
BDCA-2; 32 cycles for 18S) at 95°C for 10 s, 60°C for 10 s, and 72°C for
25 s. At each cycle, accumulation of PCR products was detected by mon-
itoring the increase in fluorescence by dsDNA-binding SYBR Green. A
dissociation/melting curve of the PCR product was constructed in the range
of 55°C to 95°C. Data were analyzed using the Bio-Rad iCycler software
and comparative threshold (Ct) method with the following formula: ?Ct ?
Ct (target, CD123 or BDCA-2) ? Ct (normalizer, 18S). Fold increase in
the expression of CD123 and BDCA-2mRNA in experimental groups com-
pared with medium control was calculated as 2?(??Ct).
Table I. Characteristics of patients included in the study
(units per liter)
Alanine aminotransferase (units
Liver biopsy performed
Ishak stage 1–2
Ishak stage 3–4
Mild (Ishak score 0–6)
Moderate (Ishak score 7–12)
43 ? 11 46 ? 1044 ? 1345 ? 8
74 ? 38 25 ? 1469 ? 4062 ? 28
86 ? 4229 ? 18 89 ? 3676 ? 31
1.96 ? 106? 0.21 ? 106
02.02 ? 106? 0.44 ? 106
6759The Journal of Immunology
by guest on June 13, 2013
The Wilcoxon nonparametric test, Bartlett’s test, and Correlation Z
score analyses in StatView (SSS Institute) program on a MacG4 com-
puter (Apple) were used.
Low IFN-? production correlates with reduced frequency
of PDCs in the peripheral blood of patients with chronic
IFN-? is a naturally occurring protein with distinct antiviral and
immunoregulatory effects (13). Healthy individuals produce IFN-?
in response to infection and stimulation with an Ag or mitogen. We
tested the hypothesis that PBMCs from patients with chronic HCV
infection may have impaired capacity to produce IFN-?. Baseline
levels of IFN-? in PBMCs from treatment-naive chronic HCV-
infected patients and normal controls were low and comparable
(Fig. 1A). Because of the unique expression of TLR9 and the ca-
pacity of PDCs to produce IFN-?, evaluation of IFN-? levels after
stimulation of PBMCs with TLR9 ligands, CpGA or HSV, is re-
flective of PDC functions (5–7, 14, 15). Upon in vitro stimulation
with TLR9 ligands, we found significantly reduced production of
IFN-? in HCV-infected patients compared with controls (CpG-A,
p ? 0.004; HSV, p ? 0.003) (Fig 1A). Such a profound defect in
IFN-? production could be explained by either functional impair-
ment or loss of PDCs. To identify the circulating PDC population,
we took advantage of the distinct surface markers, BDCA-2 and
CD123, expressed on PDCs (6–8). Using flow cytometry, we
found that patients with chronic HCV infection had a remarkable
loss of the circulating population of PDCs (determined as
BDCA2?/CD123?), compared with controls (Fig 1B). The aver-
age frequency of circulating PDCs in HCV-infected patients was
reduced to 0.12 ? 0.0.04%, from an average of 0.28 ? 0.11% seen
in controls (p ? 0.0003) (Fig. 1C).
Increased PDC apoptosis and impaired IFN-? production
correlate with active HCV infection
Cell death by apoptosis, characterized by phosphatidylserine ex-
pression on integral cellular membranes, or necrosis may account
for the loss of a cell population (16). Annexin VFITC/propidium
iodide (PI) staining revealed that PDCs isolated from healthy con-
trols exhibited low baseline apoptosis, which was increased after in
vitro stimulation with a TLR9 ligand (Fig. 2, A and B). In contrast,
freshly isolated PDCs from HCV-infected patients showed in-
creased frequency of annexin V?PDCs compared with controls
suggesting increased baseline apoptosis. We found an even higher
frequency of apoptotic PDCs from HCV-infected patients after in
vitro stimulation with a TLR9 ligand (Fig. 2, A and B). The stain-
ing of freshly isolated PDCs with trypan blue revealed no differ-
ences in membrane integrity between HCV-infected patients and
controls (data not shown), suggesting that cellular necrosis is un-
likely to account for the loss of circulating PDCs. Overall, there
was significantly higher apoptosis in the PDCs of HCV patients
compared with controls both at baseline (p ? 0.026) and after
TLR9 stimulation (p ? 0.032) (Fig. 2B). These data suggested that
PDCs from HCV patients were in vivo programmed for apoptosis.
To assess the functional capacity of PDCs, we purified
BDCA2?cells using magnetic cell sorting. Although the recovery
of PDCs from PBMCs was proportional to the low frequency of
PDCs determined by flow cytometry, there were no differences in
the levels of expression of CD123 and BDCA2 markers between
PDCs isolated from HCV patients and controls (data not shown).
Equal numbers of PDCs purified from HCV patients produced sig-
nificantly lower levels of IFN-? upon in vitro stimulation with
TLR9 ligands compared with controls (Fig. 2C). These results led
us to the conclusion that there were two components to the reduced
IFN-? production by PDCs seen in HCV-infected patients: first,
the frequency of circulating PDCs was reduced, and second, there
was an additional defect in the functional capacity of PDCs to
To determine whether PDC dysfunction correlated with ongoing
HCV infection, we tested patients who cleared the HCV virus after
IFN-?-based therapy and had no detectable viral levels (sustained
responders) and compared with those who failed to clear HCV
after therapy (nonresponders). In contrast to nonresponder patients
with detectable HCV load, sustained responders showed no dif-
ference in IFN-? production compared with HCV-naive controls
(Fig. 3A). In addition, the frequency of circulating PDCs was com-
parable between sustained responders (0.3 ? 0.22%) and controls
(0.28 ? 0.15%) but was reduced in nonresponders (0.19 ? 0.09%)
(Fig. 3B). We identified that the PDC frequency inversely correlated
with the levels of HCV core protein in the plasma of patients with
chronic HCV infection (Fig. 3C) and did not correlate with the pa-
tients’ age, viral count, or liver enzymes (data not shown). These
results suggested that ongoing viral replication and/or virus-induced
mechanisms may be responsible for the loss of PDCs in patients.
HCV core protein inhibits IFN-? production in PBMCs but not
in isolated PDCs
Based on the observation that PDC loss was associated with on-
going HCV infection and the presence of detectable HCV core
production and reduced frequency of PDCs in peripheral blood. A, PBMCs
(1 ? 107/ml) of controls (n ? 14) and treatment-naive HCV-infected pa-
tients (n ? 18) were stimulated in vitro with CpG-A (5 mM) or UV-
irradiated HSV (1 PFU/cell) for 48 h, and IFN-? production was analyzed
by ELISA. B, Freshly isolated PBMCs were stained with the indicated Abs,
fixed, and analyzed by flow cytometry, as described in Materials and Meth-
ods. Fluorescence intensity dot blots from one representative control and
one HCV-infected patient are shown. C, The frequency of PDCs in indi-
vidual controls and HCV-infected patients based on BDCA2?/CD123?
staining is shown. The horizontal bars indicate the means.
Patients with chronic HCV infection show impaired IFN-?
6760HCV CORE INHIBITS PDCs VIA MONOCYTES
by guest on June 13, 2013
protein in chronically infected patients, we focused on viral factors
that may account for the PDC defects. Among the 10 different
proteins encoded by the HCV genome, core protein has multiple
immunomodulatory effects (17). Furthermore, HCV core protein is
detectable in the peripheral blood (Fig. 3C), allowing it to interact
with immune cells outside of the liver (18). Consequently, we
and produce low IFN-?. PDCs were purified by MACS, based on BDCA-2
expression, as described in Materials and Methods, from normal controls
and treatment-naive HCV patients. A, Freshly isolated PDCs or PDCs stim-
ulated with TLR9 ligand (UV-irradiated HSV, 1 PFU/cell) for 48 h were
stained with Annexin VFITCand PI and analyzed by flow cytometry. Upper
left quadrant represents necrotic cells, upper right represents late apopto-
sis, lower right represents early apoptosis, and lower left are viable cells.
The fluorescence intensity dot blots are shown from one representative
control (top) and one patient with chronic HCV infection (bottom). B,
PDCs were cultivated, stained, and analyzed as in A. Data are shown as the
frequency of annexin V?cells from PDCs of 14 normal and 18 HCV-
infected individuals (? indicates p ? 0.05 between medium and HSV-
stimulated (TLR9) samples in controls; # indicates p ? 0.05 between me-
dium and TLR9-stimulated samples in HCV patients). C, PDC from
controls and HCV patients (1 ? 105/well) were stimulated in vitro with
CpG-A (5 mM) or UV-irradiated HSV (1 PFU/cell) for 48 h, and IFN-?
production was analyzed by ELISA (? indicates p ? 0.005 between me-
dium and CpG-A and p ? 0.001 between medium and HSV in controls; #
indicates p ? 0.025 between medium and CpG-A and p ? 0.032 between
medium and HSV in HCV patients).
Purified PDCs from HCV-infected patients are apoptotic
patients who cleared HCV infection after antiviral therapy. PBMC (1 ?
107/ml) of controls (n ? 26) and HCV patients who cleared (sustained
responders, n ? 16) or failed to clear the virus after therapy (nonre-
sponders, n ? 14) were stimulated in vitro with TLR9 ligand (CpG-A, 5
mM) for 48 h to assess IFN-? production by ELISA (A) or stained with
anti-CD123 and anti-BDCA-2 Abs and analyzed by flow cytometry (B). No
significant changes between controls and responders were observed. C, The
HCV core protein in plasma of HCV patients was quantified using Ortho
HCV core Ag ELISA. The frequency of PDCs in peripheral circulation was
plotted against the corresponding value of plasma HCV core protein. Cor-
relation z score ? ?3.402, p ? 0.026 (Bartlett test). The inset shows the
magnified picture for the HCV core values ?1000 fMol/L.
IFN-? production and frequency of PDCs is normalized in
6761The Journal of Immunology
by guest on June 13, 2013
hypothesized that HCV core protein may impair IFN-? production.
As shown in Fig. 4A, addition of rHCV core protein failed to
induce IFN-? production or affect IFN-? production in TLR9-
stimulated purified PDCs. In contrast, we found a significant re-
duction of TLR9-triggered IFN-? production in PBMCs in the
presence of HCV core protein (Fig. 4B). Our recombinant core
protein contained ?-galactosidase as a stabilizer; thus, to control
for the biological activity of the core protein, we included a ?-ga-
lactosidase control in the assay. As shown in Fig. 4B, ?-galacto-
sidase did not activate PBMCs and failed to influence TLR9-trig-
gered IFN-? production. This suggested the possibility that HCV
core protein indirectly inhibited IFN-? production by PDCs and
perhaps affected a cell population other than PDCs.
PDC function is regulated closely by the cytokine microenvi-
ronment (19); thus, we sought to evaluate whether HCV core pro-
tein-induced changes in the cytokine milieu of TLR9-stimulated
PBMCs affected IFN-? production. We have shown previously
that HCV core protein triggers cytokine production in monocytes
(12, 20). Consistent with this, HCV core protein, alone or in com-
bination with a TLR9 ligand, induced IL-10 (Fig. 4C) and TNF-?
(Fig. 4D) in PBMCs from controls and from HCV-infected pa-
tients. TLR9 stimulation alone induced minimal IL-10 or TNF-?
compared with unstimulated cells and did not alter HCV core-
induced production of these cytokines. The levels of IL-10 and
TNF-? were consistently higher in HCV-infected patients com-
pared with controls. Consistent with our previous study (12, 20),
the ?-galactosidase control did not induce IL-10 or TNF-? in
monocytes (Fig. 4, C and D). Taken together, these data implied
that in the PBMC population, HCV core protein did not affect
PDCs directly; however, it triggered production of both IL-10 and
TNF-? that, in turn, may affect IFN-? production by PDCs.
IL-10 and TNF-? inhibit TLR9-induced IFN-? production and
induce PDC apoptosis
To investigate the possibility that HCV core protein-induced IL-10
and/or TNF-? could influence IFN-? production, we stimulated
PBMCs or PDCs with rIL-10 or TNF-? in the presence or absence
of TLR9 stimulation. Addition of rIL-10 significantly inhibited
TLR9-induced IFN-? production in both PBMCs (Fig. 5A) and
PDCs (data not shown) in a dose-dependent manner. The lowest
dose of IL-10 concentration (100 pg/ml) that was comparable to
levels induced in PBMCs of HCV-infected patients (Fig. 4C) still
resulted in inhibition of IFN-? production by PDCs. Flow cytom-
etry analysis of Annexin VFITC/PI-stained cells revealed that
rIL-10 alone induced apoptosis in PDCs, and an even more dra-
matic increase in PDC apoptosis was observed when rIL-10 was
added together with a TLR9 ligand (Fig. 5B). Both the highest
dose of IL-10 (10 ng/ml) and the combination of IL-10 with a
TLR9 ligand resulted in an increase of PI?, late apoptotic cells.
Similar to the effect of IL-10, rTNF-? abolished TLR9-induced
IFN-? production in a dose-dependent manner in PBMCs (Fig.
5C) and in PDCs (data not shown). TNF-? also induced PDC
apoptosis and augmented TLR9-induced cell death (Fig. 5D).
These results demonstrated that IL-10 and TNF-? each inhibit
IFN-? production and induce apoptosis of PDCs.
Monocytes are the source of HCV core-induced cytokines that
inhibit IFN-? production by PDCs
Since rIL-10 and TNF-? could mimic the effects of HCV core
protein on TLR9-induced IFN-? production in PBMCs, we inves-
tigated whether neutralization of IL-10 and TNF-? could eliminate
the inhibitory effect of HCV core protein on IFN-? production. As
shown in Fig. 6A, anti-IL-10- and anti-TNF-?-neutralizing Abs
each partially blocked the inhibitory effect of HCV core protein on
IFN-? production by PBMCs. Furthermore, the combination of
PBMCs but not in purified PDC population. Purified PDCs (A) (1 ? 106/
ml) or PBMCs (B) (1 ? 107/ml) were stimulated with TLR9 ligand (HSV,
1 PFU/cell), HCV core (22,500 fM), ?-galactosidase (5 ?g/ml) or their
combination, as indicated. Forty-eight hours later the production of IFN-?
in cell-free culture supernatants was determined using ELISA. Mean ? SE
from five controls and six HCV-infected treatment-naive patients are
shown as nanograms per milliliter. C and D, PBMCs were stimulated as
described above. Culture supernatants were analyzed for IL-10 (C) and
TNF-? (D) production in ELISA. Mean ? SE from five controls and six
HCV-infected patients is shown. (C, ? indicates p ? 0.023 between me-
dium and HCV core; # indicates p ? 0.031 between TLR9 ligand and
TLR9?HCV core in controls; ¶ indicates p ? 0.021 between medium and
HCV core; and § indicates p ? 0.028 between TLR9 ligand and TLR9?
HCV core in HCV-infected patients). (D, ? indicates p ? 0.016 between
medium and HCV core and # indicates p ? 0.036 between TLR9 ligand
and TLR9?HCV core in controls; ¶ indicates p ? 0.028 between medium
and HCV core; and § indicates p ? 0.038 between TLR9 ligand and
TLR9?HCV core in HCV patients).
HCV core inhibits TLR9-triggered IFN-? production in
6762HCV CORE INHIBITS PDCs VIA MONOCYTES
by guest on June 13, 2013
anti-IL-10- and anti-TNF-?-neutralizing Abs resulted in an addi-
tive effect and totally blocked IFN-? inhibition by HCV core pro-
tein. To confirm the specificity of the neutralizing Abs, we mim-
icked the HCV core action by adding rIL-10, TNF-?, and their
combination to TLR9-stimulated PBMCs and demonstrated that
anti-IL-10- and anti-TNF-?-neutralizing Abs could prevent the
IL-10- and TNF-?-induced inhibition of IFN-? production, respec-
tively. These data confirmed that both IL-10 and TNF-? are im-
plicated in HCV core-mediated inhibition of IFN-? production.
PBMCs represent a heterogeneous population consisting of lym-
phocytes, NK cells, monocytes, and circulating DCs. Of those,
lymphocytes produce negligible amounts of IL-10, whereas mono-
cytes are the main producers of IL-10 and TNF-? due to their
numeric prevalence over DCs. Thus, we tested the hypothesis that
monocytes are the source of HCV core-induced IL-10 and TNF-?
that inhibit IFN-? production by PDCs. Magnetic bead separation
of CD14?cells from PBMCs resulted in a 100% pure CD14?
population and a ?90% pure CD14?population (data not shown).
Depletion of CD14?cells did not affect TLR9-induced production
of IFN-?, but it fully prevented the HCV core-induced inhibition
of IFN-? production in TLR9 ligand-stimulated PBMCs (Fig. 6B).
The purified CD14?cell population from both HCV-infected pa-
tients and normal controls produced IL-10 (Fig. 6C) and TNF-?
(Fig. 6D) in response to HCV core and LPS, a TLR4/CD14 ligand,
used as a positive control for monocyte activation. These results
confirmed that CD14?monocytes are the primary target of HCV
core protein in induction of IL-10 and TNF-? that inhibit IFN-?
production by PDCs. HCV core protein inhibited TLR9-triggered
IFN-? production in a dose-dependent manner (Fig. 6E), and the
lowest dose of HCV core protein that affected IFN-? (?4500 fM)
was close to the range of HCV core protein levels detected in
plasma of patients with chronic HCV infection (Fig. 3C).
The critical role of monocytes in mediation of HCV core-induced
inhibition of IFN-? production was further investigated in cocultures
of isolated PDCs with monocytes or T lymphocytes. While TLR9
stimulation induced IFN-? production in PDCs in the presence of
either monocytes or T cells, inhibition of IFN-? production by HCV
core protein occurred only when PDCs were cocultured with mono-
cytes and not with T cells (Fig. 7A). Both TNF-? and IL-10 were
induced by HCV core protein in the monocyte-PDC but not in the T
cell-PDC cocultures, further pointing to the role of monocyte-derived
cytokines in inhibition of IFN-? production by PDCs (Fig. 7, B and
C). We and others have reported previously that HCV core protein
triggers monocyte activation via TLRs, in particular by membrane
stimulated in vitro with TLR9 ligand (HSV, 1 PFU/cell) with or without rIL-10 (A) or TNF-? (C), as indicated. After 48 h, IFN-? production was analyzed
in culture supernatants using ELISA. ?, p ? 0.05 compared to TLR9 ligand alone. B and D, PDCs from control donors were cultivated in vitro in the absence
(medium) or presence of IL-10 or TNF-? (concentrations as indicated), TLR9 ligand (HSV, 1 PFU/cell), or a combination of TLR9 ligand plus IL-10 or
TNF-? (concentrations as indicated) for 48 h. PDCs were stained with Annexin VFITCand PI and analyzed by flow cytometry. Fluorescence intensity dot
blots from one representative control of n ? 3 with similar results are shown. Increased annexin V staining indicates apoptosis.
IL-10 and TNF-? impair IFN-? production and induce PDC apoptosis. A and C, PBMCs (1 ? 107/ml) from normal controls (n ? 4) were
6763The Journal of Immunology
by guest on June 13, 2013
expressed TLR2 (20) and TLR4 (21). In the present study, we found
that anti-TLR2, but not anti-TLR4, can partially restore the HCV
core-mediated inhibition of TLR9-triggered IFN-? production in
PBMCs (Fig. 7D). The specific activity of the anti-TLR2- and
anti-TLR4-blocking Abs was indicated by inhibition proteoglycan
(TLR2 ligand)- and LPS (TLR4 ligand)-mediated TNF-? produc-
tion in monocytes, respectively (Fig. 7E). These data suggest that
TLR2 plays a critical role in monocyte activation and is implicated
in the inhibitory effect of HCV core on IFN-? production
To examine whether HCV core protein and the cytokine milieu
played a role in PDC functional impairment, we analyzed a cohort
of patients with NASH, an inflammatory liver disease of nonviral
etiology (22). We found that the frequency of PDCs in the periph-
eral circulation (Fig. 8A) and the production of IFN-? (Fig. 8B),
TNF-? (Fig. 8C), and IL-10 (Fig. 8D) in PBMCs were comparable
between patients with NASH and controls. Furthermore, similar to
controls, HCV core protein reduced the TLR9-triggered IFN-?
production in PBMCs of NASH patients (Fig. 8B), suggesting that
HCV core protein can impair PDCs functions.
Finally, we considered the hypothesis that the apparent “loss” of
PDC numbers in the blood of patients with chronic HCV infection
could be due to PDC homing to the liver, the primary site of HCV
infection. We identified that the levels of mRNA coding for the
PDC markers (5–8), CD123 (Fig. 9A) and BDCA-2 (Fig. 9B),
were significantly higher in the livers of patients with HCV infec-
tion compared with noninfected controls or patients with
suggesting that chronic HCV infection may lead to PDC
Although various host- and virus-derived factors can account for
viral persistence during the course of chronic hepatitis C infection,
the complexity of the immune defects and the interactions between
the virus and different immune cell types remains to be fully un-
derstood. In the present study, we report that patients with chronic
HCV infection have reduced capacity to produce IFN-? after in
vitro stimulation of PDCs. Our studies delineated at least three
reasons for the reduced IFN-? production in these patients. First,
we found a loss of circulating PDCs due to apoptosis. Second,
there was reduced capacity of existing circulating PDCs to produce
IFN-?. Third, we identified increased PDC homing to the liver in
HCV infection. We also demonstrated that reduced circulating
PDC frequency correlated with increased plasma levels of HCV
core protein. Furthermore, we found that in PBMC HCV core pro-
tein triggered monocyte-derived IL-10 and TNF-? production.
These cytokines, in turn, led to PDC apoptosis and impaired pro-
duction of IFN-?, thus closely resembling the PDC defects seen in
chronically HCV-infected patients.
PDCs produce large amounts of IFN-? upon viral infection (7,
8). We found that PDCs of HCV-infected patients had reduced
IFN-? production capacity upon TLR9 stimulation with a syn-
thetic ligand, CpG-A, or a natural ligand, UV-inactivated HSV
(6–8, 14, 15). These data are in agreement with previous publi-
cations from Murakami et al. (23) and Wertheimer et al. (24), and
in contradiction with a report from Longman et al. (25), possibly
due to the differences in the approaches taken to evaluate the IFN-
?-producing capacity of PDCs. IFN-? is detrimental for establish-
ing an effective link between the innate and adaptive immunity via
up-regulation of IFN-? in CD4?and CD8?T cells and NK cells,
modulation of T cell responsiveness to IL-12-induced secretion of
IFN-?, and subsequent drive of a Th1 response (7, 26). Indeed,
multiple immune defects have been described in patients with
by PDCs. A, PBMCs from normal individuals (n ? 5) were seeded at 1 ?
107/ml and preincubated with isotype control (10 ?g/ml), anti-IL-10 (10 ?g/
ml), anti-TNF-? (10 ?g/ml), or a combination of anti-IL-10 plus anti-TNF-?
(10 ?g/ml each) Abs for 30 min at room temperature. The cells were then
stimulated with UV-irradiated TLR9 ligand (HSV, 1 PFU/cell) or combination
of TLR9 ligand with HCV core (22,500 fM), or IL-10 (0.1 ng/ml), or TNF-?
(0.01 ng/ml), or IL-10 (0.1 ng/ml) plus TNF-? (0.01 ng/ml), as indicated.
Culture supernatants were harvested 48 h later, and IFN-? production was
analyzed in ELISA. The ? represents p ? 0.05 compared with TLR9 stimu-
lation. B, PBMCs (1 ? 107/sample/ml) and corresponding numbers of CD14-
depleted PBMCs from four normal controls were stimulated with TLR9 ligand
(22,500 fM) for 48 h. Culture supernatants were analyzed for IFN-? produc-
tion; data shown as mean ? SE ng/ml. “ns” indicates no significant changes
between controls and HCV-infected patients. C and D, CD14?cells (1 ?
106/ml) were purified from PBMCs as described in Materials and Methods
and stimulated with HCV core (22,500 fM) for 48 h, then analyzed for IL-10
(LPS, 1 ?g/ml) was used as a positive control for monocyte function; ?-ga-
lactosidase was included as a negative control for HCV core protein. Data
from three controls and five HCV-infected patients are shown, as mean ? SE
pg/ml. ns indicates no significant changes between controls and HCV-infected
patients. E, Human PBMCs were stimulated with TLR9 ligand (HSV, 1 PFU/
cell) and increasing concentrations of rHCV core protein as indicated for 48 h.
IFN-? production in cell-free supernatants was analyzed in ELISA (n ? 4).
The ? indicates p ? 0.05 compared with TLR9 ligand.
Monocyte-derived IL-10 and TNF-? inhibit IFN-? production
6764HCV CORE INHIBITS PDCs VIA MONOCYTES
by guest on June 13, 2013
chronic HCV infection that may be linked to reduced IFN-? pro-
duction, including insufficient response of CTLs, low activity of
NK cells, and production of Abs with low neutralizing capacity (2,
13, 27–29). It has been reported that IFN-? is a potent survival
factor of PDCs (30, 31); thus, internal deficit of IFN-? may favor
a self-destructive loop for loss of PDCs in HCV-infected patients.
PDCs in the presence of monocytes but not T lymphocytes. BDCA-2?
PDCs from healthy donors (1 ? 105/well) and syngeneic monocytes (1 ?
106/well, u) or T lymphocytes (1 ? 106/well, f) were stimulated in vitro
HCV core impairs TLR9-induced IFN-? production in
with TLR9 ligand (CpG-A, 5 mM) with or without HCV core protein
(22,500 fM) for 48 h at 1:10 PDC:monocyte or PDC:T cell ratio. The
production of IFN-? (A), TNF-? (B), and IL-10 (C) was analyzed in
ELISA (n ? 3). D, Human PBMCs were incubated with indicated Abs for
30 min at room temperature, then stimulated with TLR9 ligand (HSV, 1
PFU/cell) with or without rHCV core protein (22,500 fM) as indicated for
48 h. IFN-? production in cell-free supernatants was analyzed in ELISA
(n ? 3). The ? indicates a p ? 0.05 compared with TLR9 ligand plus
corresponding control Abs; the ¶ indicates a p ? 0.05 compared with
TLR9 ligand. E, Human monocytes were incubated with indicated Abs for
30 min at room temperature and then stimulated with TLR2 (proteoglycan
(PGN), 1 ?g/ml) or TLR4 (LPS, 100 ?g/ml) ligands for 16 h. TNF-?
production in cell-free supernatants was analyzed in ELISA (n ? 3). The
? indicated a p ? 0.05 compared with corresponding control Abs.
patients is comparable to controls. A, The frequency of PDCs
(BDCA2?CD123?) in individual controls and NASH patients is shown.
The horizontal bars indicate the means; ns indicate no significant changes.
B–D, PBMCs (1 ? 107/sample/ml) from normal controls (n ? 4) or NASH
patients (n ? 6) were stimulated with TLR9 ligand (HSV, 1 PFU/cell)
alone or with a combination of TLR9 ligand plus HCV core (22,500 fM).
Culture supernatants were analyzed for IFN-? (B), TNF-? (C), or IL-10
(D) production in specific ELISA; data are shown as mean ? SE ng/ml.
The frequency of PDCs and cytokine production in NASH
6765The Journal of Immunology
by guest on June 13, 2013
Our results suggested that circulating PDCs have increased base-
line and TLR9-induced apoptosis in patients with chronic HCV
infection. Reduced absolute numbers of the circulating PDCs were
reported previously in patients with chronic HCV compared with
controls (23–25). A HCV escape mechanism from IFN-?-medi-
ated antiviral effects would be most important in the liver, where
HCV replicates and most of the virus and the virus-derived pro-
teins are produced. High IFN-? production has been described
during the acute phase of HCV infection (32, 33); however, reports
are contradictory with regards to chronic HCV infection (8–11). In
the present study, we identified increased mRNA levels of the PDC
markers, BDCA2 and CD123, suggesting that PDCs can be re-
cruited to the liver in patients with chronic HCV infection. Con-
sistent with this notion, CD80?, CD83?, and CD86?cells with
DC morphology were identified in livers of HCV-infected patients
(34). The functional capacity of those PDCs in the liver remains to
be evaluated. While our data shows PDC dysfunctions, reduced
Ag-specific T cell activation and Ag presentation capacity of
myeloid and monocyte-derived DCs may further contribute to
the loss in virus-specific immune activation in chronic HCV
infection (12, 35, 36).
Our observation that only patients with detectable levels of
HCV, and not sustained responders or patients with NASH, en-
countered loss of PDCs and impaired IFN-? production capacity
suggested a direct role for the virus in the loss and functional
impairment of PDCs. HCV encodes a long polyprotein that is
cleaved into at least 10 known proteins (37). Core protein, the first
to be cleaved from the viral polyprotein, has a distinct immuno-
regulatory capacity. In agreement with previous studies (18, 38),
we found that HCV core protein was found in the peripheral blood
of all analyzed HCV-infected patients. Furthermore, we identified
an inverse relationship between plasma core protein levels and
circulating PDC frequency in patients with chronic HCV infection.
Our studies revealed that HCV core protein induced IL-10 and
TNF-? production in monocytes, and these cytokines in turn re-
sulted in PDC apoptosis and impaired capacity of PDCs to produce
HCV core protein can interact with multiple cellular factors;
regulate expression of cellular genes and control signaling path-
ways in different cell types, including DCs (reviewed in Refs. 17
and 37). We have reported previously that monocytes recognize
HCV core protein via TLR2 and TLR2 activation triggers intra-
cellular signaling pathways to induce TNF-? (20). Recently, TLR4
was also implicated in cellular activation by HCV core protein
(21). In the present study, we found that anti-TLR2, but not anti-
TLR4, Abs partially prevented the inhibitory effect of HCV core
protein on TLR9-triggered IFN-? production, thus suggesting that
core protein, via TLR2, plays a role in modulation of PDC func-
tions. We have shown previously that the intracellular signaling
pathways triggered by HCV core involved in activation of NF-?B,
AP-1, and the MAPKs in induction of IL-10 and TNF-? (20). Both
IL-10 and TNF-? production were induced in macrophages by
HCV core protein at concentrations comparable to HCV core pro-
tein levels found in the sera of HCV-infected individuals. It is also
likely that macrophages and monocytes in the liver may be ex-
posed to even higher concentrations of HCV core protein than the
circulating levels. Furthermore, monocytes of patients with
chronic HCV infection produced higher levels IL-10 and TNF-?,
and increased levels of these cytokines were reported in plasma
from HCV-infected patients compared with controls (39, 40). In
the present study, we demonstrated that Ab neutralization of IL-10
and TNF-? restored IFN-? production in TLR9 plus HCV core-
stimulated PBMCs. Furthermore, rIL-10 and TNF-? inhibited both
TLR9-induced IFN-? production and PDC apoptosis in a dose-
dependent manner. This observation is in agreement with previous
publications showing that the microenvironment, including the
presence of cytokines and chemokines, is crucial for PDC function
(19, 41–43). ILs 3, 4, 7, and 15 promote IFN-? production by
virus-infected cells whereas TNF-? and IL-10 inhibit it (19). We
identified that CD14?monocytes are the main targets of HCV
core. Although HCV-specific CD4 and CD8 T cells with Ag-spe-
cific IL-10 and/or TNF-? production have been reported in various
studies (44, 45), our coculture experiments revealed that IFN-?
production was affected only in PDC/monocyte but not in PDC/T
cell cocultures. This may be related to TLR2 recognition of core
protein because TLR2 on monocytes has independent recognition
and stimulatory function, while in T cells, TLR2 requires TCR
costimulation for cell activation (46, 47).
Under normal conditions, both TNF-? and IL-10 limit overpro-
duction of IFN-? by activated PDCs during resolution of viral
infections (19). Our data suggest that HCV can steal this protective
mechanism to achieve a destructive effect on PDCs by inducing
TNF-? and IL-10 in monocytes. It has been suggested previously
that IL-10 may reduce PDC viability, but the mechanism of IL-
10-induced PDC death is unknown to date (41). We dissected the
mechanism of IL-10- and TNF-?-induced PDC death and found
that both IL-10 and TNF-? induced PDC apoptosis. Although this
effect is expected for TNF-?, a known trigger of apoptosis in dif-
ferent cell types, the observation of PDC apoptosis induction by
IL-10 is novel. A recent report by Marra et al. (48) showed that
IL-10 induces regulatory T cell apoptosis by up-regulation of the
membrane form of TNF-?. Consequently, it is not surprising that
we found an additive protection by anti-IL-10 plus anti-TNF-?
Abs on PDC function.
We showed that chronic HCV infection induces cytokine
changes and leads to PDC dysfunction as measured by IFN-? pro-
duction and apoptosis; thus, one could suspect that HCV may in-
duce global immune suppression. In support of this notion, recent
data from El-Sarag et al. (49) from a case-control study showed
CD123 and BDCA-2 in the liver. The levels of RNA coding for CD123 (A)
and BDCA-2 (B) was evaluated by PCR in livers of controls (n ? 3),
NASH patients (n ? 6), and HCV-infected, treatment-naive patients (n ?
28). The ? shows p ? 0.05 compared with controls.
HCV-infected patients have elevated levels of PDC markers
6766 HCV CORE INHIBITS PDCs VIA MONOCYTES
by guest on June 13, 2013
that patients with HCV had a significantly higher prevalence of
other blood-borne virus infections, including HIV, hepatitis B,
CMV, as well as cryptococcus, tuberculosis, and sexually trans-
mitted diseases. This suggests that patients with chronic HCV in-
fection may indeed have signs of immunosuppression, leading to
increased rate of other infections. Indeed, impaired IFN-? produc-
tion and depletion of PDCs are common in viral infections, in-
cluding HIV, human T cell leukemia virus type 1, and hepatitis B
virus (50–54); however, the mechanisms of PDC depletion may
not be identical in all viral infections. While direct cytolytic effect
on PDCs is possible for retroviruses (HIV-1 and human T cell
leukemia virus type 1), it is less probable for hepadnaviruses such
as HBV and flaviviruses such as HCV. Distinct patterns of IFN-?
production were described in patients infected with HIV-1 alone,
HCV, or HIV-1/HCV coinfection (54), suggesting that different
mechanisms account for the PDC functional impairments. Consis-
tent with this, we found that unlike HCV core protein, HIV p24 or
HBV core proteins did not inhibit IFN-? production in PBMCs
(data not shown). We found that HCV used alternative tactics by
acting on monocytes and affecting PDCs indirectly via monocyte-
derived mediators. While our results offer a mechanistic explana-
tion for HCV-induced PDC apoptosis via monocyte-mediated cy-
tokine production, we cannot rule out the possibility that the
increased baseline apoptosis found in PDCs of patients with
chronic HCV infection would be due to a direct action of the virus
on PDCs. Recent reports show the existence of extrahepatic sites
of HCV replication, including PDCs (55). Interestingly, in vivo
infection with HSV resulted in unresponsiveness of PDCs to sub-
sequent in vitro rechallenge (56). Thus, we cannot rule out the
possibility that HCV may directly affect PDCs in vivo, resulting in
their apoptosis and unresponsiveness to subsequent TLR challenge
in vitro, as found in our experiments.
In conclusion, we demonstrate that patients chronically infected
with HCV have a profound defect in PDC frequency and IFN-?
production capacity. Our results suggest that HCV patients’ PDCs
are in vivo programmed for apoptosis and have diminished IFN-
?-producing capacity. We identified that HCV core-induced IL-10
and TNF-? mediated these PDC defects, and removal of cytokine-
producing monocytes normalized PDC IFN-? production even in
the presence of HCV core protein. Our study indicates that viral-
induced mechanisms of PDC loss and IFN-? production defects
are likely to contribute to chronic viral persistence and may pro-
vide mechanistic explanations for the therapeutic benefits of IFN-?
in HCV infection.
We are grateful to Dr. Savant Mehta, Dr. Lawton Shick, Down Bombard,
R.N., DonnaGiansiracusa, R.N.,
D’Agostino, N.P., and Kathy Coleman, N.P., for their help with patient
recruitment. The cooperation from our patients and blood donors at Uni-
versity of Massachusetts Medical School is greatly appreciated. We thank
Dr. Raymond Welsh (University of Massachusetts Medical School) for
HSV KOS-1 stock, Dr. Gunther Hartmann (University of Munich) for
helpful discussions, and UMass Flow Cytometry Core Facility for excellent
support with flow cytometry.
The authors have no financial conflict of interest.
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6768HCV CORE INHIBITS PDCs VIA MONOCYTES
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