Article

Hepatitis C Virus Inhibits Cell Surface Expression of HLA-DR, Prevents Dendritic Cell Maturation, and Induces Interleukin-10 Production

Departments of Internal Medicine, Saint Louis University, St. Louis, Missouri, USA.
Journal of Virology (Impact Factor: 4.44). 05/2008; 82(7):3320-8. DOI: 10.1128/JVI.02547-07
Source: PubMed

ABSTRACT

Hepatitis C virus (HCV) chronic infection is characterized by low-level or undetectable cellular immune responses against HCV antigens. HCV proteins have been shown to affect various intracellular events and modulate immune responses, although the precise mechanisms used to mediate these effects are not fully understood. In this study, we have examined the effect of HCV proteins on the modulation of major histocompatibility complex (MHC) class II expression and other functions important for antigen presentation in humans. Expression of an HCV(1-2962) genomic clone (HCV-FL) in human fibrosarcoma cells (HT1080) inhibited gamma interferon (IFN-gamma)-induced upregulation of human leukocyte antigen-DR (HLA-DR) cell surface expression. Furthermore, inhibition of promoter activities of MHC class II transactivator (CIITA), IFN-gamma-activated site (GAS), and HLA-DR was observed in IFN-gamma-inducible HT1080 cells expressing HCV-FL by in vitro reporter assays. Exposure of human monocyte-derived dendritic cells (DCs) to cell culture-grown HCV (HCVcc) genotype 1a (clone H77) or 2a (clone JFH1) significantly inhibited DC maturation and was associated with the production of IL-10. Furthermore, DCs exposed to HCVcc were impaired in their functional ability to stimulate antigen-specific CD4-positive (CD4(+)) and CD8(+) T-cell responses. Taken together, our results indicated that HCV can have direct and/or indirect inhibitory effects on antigen-presenting cells, resulting in reduction of antigen-specific T-cell activation. These effects may account for or contribute to the low overall level of immunogenicity of HCV observed in chronically infected patients.

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JOURNAL OF VIROLOGY, Apr. 2008, p. 3320–3328 Vol. 82, No. 7
0022-538X/08/$08.000 doi:10.1128/JVI.02547-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Hepatitis C Virus Inhibits Cell Surface Expression of HLA-DR,
Prevents Dendritic Cell Maturation, and Induces
Interleukin-10 Production
Kousuke Saito,
1
Malika Ait-Goughoulte,
1
Steven M. Truscott,
1
Keith Meyer,
1
Azra Blazevic,
1
Getahun Abate,
1
Ratna B. Ray,
1,2
Daniel F. Hoft,
1,3
and Ranjit Ray
1,3
*
Departments of Internal Medicine,
1
Pathology,
2
and Molecular Microbiology & Immunology,
3
Saint Louis University,
St. Louis, Missouri
Received 28 November 2007/Accepted 10 January 2008
Hepatitis C virus (HCV) chronic infection is characterized by low-level or undetectable cellular immune
responses against HCV antigens. HCV proteins have been shown to affect various intracellular events and
modulate immune responses, although the precise mechanisms used to mediate these effects are not fully
understood. In this study, we have examined the effect of HCV proteins on the modulation of major histocom-
patibility complex (MHC) class II expression and other functions important for antigen presentation in
humans. Expression of an HCV
1–2962
genomic clone (HCV-FL) in human fibrosarcoma cells (HT1080) inhib-
ited gamma interferon (IFN-)-induced upregulation of human leukocyte antigen-DR (HLA-DR) cell surface
expression. Furthermore, inhibition of promoter activities of MHC class II transactivator (CIITA), IFN--
activated site (GAS), and HLA-DR was observed in IFN--inducible HT1080 cells expressing HCV-FL by in
vitro reporter assays. Exposure of human monocyte-derived dendritic cells (DCs) to cell culture-grown HCV
(HCVcc) genotype 1a (clone H77) or 2a (clone JFH1) significantly inhibited DC maturation and was associated
with the production of IL-10. Furthermore, DCs exposed to HCVcc were impaired in their functional ability to
stimulate antigen-specific CD4-positive (CD4
) and CD8
T-cell responses. Taken together, our results
indicated that HCV can have direct and/or indirect inhibitory effects on antigen-presenting cells, resulting in
reduction of antigen-specific T-cell activation. These effects may account for or contribute to the low overall
level of immunogenicity of HCV observed in chronically infected patients.
Hepatitis C virus (HCV) infection is one of the major causes
of chronic liver disease worldwide. In the early phase of acute
infection, HCV continues to replicate in the liver, overcoming
innate and acquired immunity in a majority of infected hu-
mans. Sustained, vigorous, and multiepitope-specific CD4-pos-
itive (CD4
) and CD8
T-cell responses are essential for
spontaneous HCV clearance (26). Infected cells display pep-
tides derived from viral antigens bound to major histocompat-
ibility complex (MHC)/human leukocyte antigen (HLA) re-
ceptors at the cell surface and are recognized by T cells.
Generation of HCV-specific CD4
T cells occurs after stimu-
lation by MHC class II/viral peptide complexes and facilitates
the induction of HCV-specific CD8
T-cell responses and op-
timal antibody production. CD8
T cells recognize and attack
infected cells expressing viral peptides in the context of MHC
class I surface molecules.
Dendritic cells (DCs) are important for the initiation of T-cell
responses to foreign antigens. The antigen uptake and presenta-
tion capacities of DCs enable them to prime and activate naı¨v e T
cells. Immature DCs are optimally phagocytic and pinocytose
antigens. However, DCs must be activated to mature before serv-
ing as efficient antigen-presenting cells (APCs). Chronic HCV
infection has been shown to affect the allostimulatory function of
DCs (3). Monocyte-derived DCs from patients with chronic HCV
infection do not respond to maturation stimuli, maintaining their
immature phenotype (1). The presence of HCV genomic se-
quences in DCs could be documented for HCV carriers (4),
although DCs may not support HCV replication. DC-SIGN and
DC-SIGNR are two closely related membrane-associated C-type
lectins. DC-SIGN is expressed on some DCs, while DC-SIGNR is
localized to certain endothelial cell populations, including hepatic
sinusoidal endothelial cells. Interactions of HCV envelope glyco-
protein E2 with DC-SIGN and DC-SIGNR may contribute to the
capture and delivery of virus to the liver by DCs (29, 39).
Several HLA polymorphisms are associated with viral clear-
ance or persistence (21, 34, 35). The HLA-DR molecules are
pivotal for the adaptive immune system, as they guide the devel-
opment and activation of CD4
T helper cells (41, 42). This
complex gene expression profile is controlled almost exclusively
by the CIITA, a global regulator for the expression of genes
involved in antigen presentation (10, 30). Differential activation of
independent promoters that drive expression of the gene encod-
ing CIITA ultimately determines the exquisitely regulated pattern
of MHC gene expression. Transcription of CIITA is driven by a
large regulatory region that contains four distinct promoters
known as pI, pII, pIII, and pIV (36). Cytokine regulation by
CIITA is complex and may reflect both direct and indirect mech-
anisms of T-cell development and differentiation (37). The pro-
moter pIV is induced by gamma interferon (IFN-), and a con-
served 300-bp promoter-proximal region is sufficient for the
activation. This region contains three regulatory elements, GAS,
* Corresponding author. Mailing address: Division of Infectious
Diseases & Immunology, Center for Vaccine Development, Edward
A. Doisy Research Center, 1100 S. Grand Blvd., 8th Floor, St. Louis,
MO 63104. Phone: (314) 977-9034. Fax: (314) 771-3816. E-mail: rayr
@slu.edu.
Published ahead of print on 23 January 2008.
3320
Page 1
an E box, and an IFN-regulatory factor element (IRF-E), all of
which are required for the induction of pIV. The main function of
HLA-DR molecules encoded by one of the three MHC class II
alleles is to present processed antigens which are derived primar-
ily from exogenous sources to CD4
T lymphocytes (22). There
-
fore, HLA-DR molecules are critical for the initiation of the
antigen-specific immune response. Constitutive expression of
MHC class II molecules is confined to professional APCs of the
immune system, while MHC class II molecule expression can be
induced by a variety of immune regulators in nonprofessional
APCs.
MHC class II proteins are important for the initiation of
immune responses and are essential for specific recognition
of foreign antigens by the immune system. Thus, an interaction
of DCs with HCV particles may play an important role in
immunopathogenesis. In this study, we have examined the role
of HCV proteins and infectious HCV in suppression of APC
function/T-cell activation. IFN- is the most potent inducer of
MHC class II expression in many cell types (8). Our results
indicate that cells endogenously expressing HCV proteins from
an HCV
1-2962
(FL) construct or those exposed to cell culture-
grown HCV (HCVcc) perturb HLA-DR cell surface expres-
sion. The inhibition of MHC class II cell surface expression
was evident on IFN--induced HT1080 cells and on profes-
sional antigen-presenting monocyte-derived DCs. A deficiency
in expression of HLA-DR molecules was further observed to
be associated with the negative regulation of CIITA, GAS, and
the HLA-DR promoters at the transcriptional level, induction
of interleukin-10 (IL-10) in DCs, and inhibition of DC-medi-
ated antigen-specific T-cell activation.
MATERIALS AND METHODS
Cells and transfections. The human fibrosarcoma epithelial-like HT1080 cell
line was a gift from George Stark (Cleveland Clinic Foundation, Cleveland, OH).
HT1080 cells were transfected with pCI-neo-HCV
1–2962
plasmid DNA (HCV-
FL) from genotype 1a (clone H77) by use of Lipofectamine (Life Technologies,
Inc., Rockville, MD). Stable cell colonies were selected using G418 and pooled
to avoid artifactual results from clonal variation, as previously described (5).
Parental HT1080 cells were used in parallel as a control. HT1080 cells, and their
transfected derivatives, were maintained in Dulbecco’s modified Eagle medium
containing 10% fetal calf serum with a lower dose of the selection antibiotic (1
g of puromycin/ml or 400 g of G418/ml).
Generation of cell culture-grown HCV. HCV genotype 1a (clone H77; HCV1)
was grown in immortalized human hepatocytes (IHH) as recently described (25).
Virus growth was measured from cell culture supernatant filtered through a
0.45-um-pore-size cellulose acetate membrane (Nalgene, Rochester, NY) by
fluorescent focus-forming assay from serial dilutions. HCV titer was calculated as
\710
5
focus-forming units/ml. HCV genotype 2a (clone JFH1; HCV2) was
grown in Huh-7.5 cells as previously described (25).
Exposure of DCs to cell culture-grown HCV. Immature DCs were prepared
from human monocytes treated with granulocyte-macrophage colony-stimulating
factor and IL-4 for 6 days (protocol approved by the Internal Review Board,
Saint Louis University), as described earlier (48). Immature DCs (1 10
5
/ml) in
RPMI 1640 supplemented with 10% fetal bovine serum and 2 mM
L-glutamine
were seeded in plastic tubes. The next day, DCs were exposed for 6 h to 10
4
focus-forming units of HCVcc or conditioned medium (CM) from mock-infected
hepatocytes as a negative control and incubated for 16 h. Subsequently, a mat-
FIG. 1. Inhibition of HLA-DR expression by HCV polyprotein in
IFN--treated HT1080 cells. (A and B) Mock-transfected HT1080
cells (A) or IFN--treated cells (B) were used as a negative and
positive controls, respectively, for assays of inducible HLA-DR expres-
sion. (C to F) HT1080 cells transfected with HCV core or HCV-FL
were incubated in the absence (C and E) or presence (D and F) of
IFN- (500 U/ml) for 48 h. Cells were subjected to FACS analysis of
surface expression of HLA-DR using specific antibodies. Similar re-
sults of percentages of cells positive for HLA-DR expression (within
2% variations) were observed in triplicate experiments.
FIG. 2. Expression of HCV-FL downregulates CIITA, GAS, and
HLA-DR promoter activities. HT1080 cells were transfected with plas-
mid DNA containing CIITA (A), GAS (B), or HLA-DR (C) promoter
with a luciferase reporter construct together with empty vector (con-
trol), HCV core, or HCV-FL from genotype 1a with Lipofectamine
2000 (Invitrogen). After 48 h of transfection, cells were incubated with
IFN- (500 U/ml) for 24 h, and luciferase gene expression was ana-
lyzed. Results represent triplicate analyses and are shown with stan-
dard errors.
V
OL. 82, 2008 INHIBITORY EFFECTS OF HCV ON ANTIGEN-PRESENTING CELLS 3321
Page 2
3322
Page 3
uration cocktail (MC) (containing a final concentration of 2 ng/ml IL-1, 1,000
U/ml IL-6, 10 ng/ml tumor necrosis factor alpha, and 1 g/ml prostaglandin E2
[PGE2]) was added and the DC cultures were incubated for 48 h more to induce
maturation. IL-10 was added in culture as a negative control for inhibition of DC
maturation. DCs were subjected to fluorescence-activated cell sorter (FACS)
analysis for phenotypic characterization.
Flow cytometry. Cell surface expression of costimulatory molecules or
HLA-DR was quantified by flow cytometry. Transfected HT1080 cells or DCs
exposed to HCVcc were grown in six-well plates (5 10
6
cells) or plastic tubes
(1 10
5
cells), respectively. Cells were treated with 0.1 mM EDTA for detaching
and harvesting, washed with phosphate-buffered saline (PBS), and stained with
fluorochrome-tagged antibodies to HLA-DR peridinin-chlorophyll-protein com-
plex, CD80 fluorescein isothiocyanate, CD14 Pac Blue, CD40 allophycocyanin,
CD83 phycoerythrin, and CD3 Alexa 700 (BD Pharmingen, San Diego, CA). For
staining, cells were incubated with 10 l of monoclonal antibody in a total
volume of 50 l (2% fetal bovine serum –PBS) for 30 min at 4°C in the dark,
washed, and resuspended in 250 l of PBS containing 1% paraformaldehyde.
Cells were gated according to their size (forward light scatter) and granularity
(side light scatter) using a Guava Easysite (Guava Technology, Hayward, CA) or
Becton Dickinson flow cytometer. Surface marker expression on gated cells was
analyzed using FlowJo (Tree Star) and CellQuest (BD Immunocytometry Sys-
tems) software. The Statistica program was used for analyses of variations.
Luciferase reporter assay. HT1080 cells (1 10
6
) were seeded 24 h before
transfection. Cells were transfected with 1 g of CIITA or DRA promoter in a
luciferase reporter construct (38) (kindly provided by Jenny P.-Y. Ting). The
GAS-luciferase construct (Stratagene) was also used in this study. Cells were
transfected together with reporter construct and 1 g HCV protein expression
plasmid or empty vector as a negative control. At 24 h after transfection, 500
U/ml IFN- was added to the cells and incubated for another 24 h. Cells were
detached with PBS containing 0.1 mM EDTA, harvested, and lysed with RLU
lysis buffer (Promega, Madison, WI) by incubation for 30 min at room temper-
ature. After clarification by centrifugation, the supernatant was subjected to a
luciferase reporter assay using a luminometer (Opticomp II; MGM Instruments).
A cytomegalovirus–-galactosidase plasmid construct was used for transfection
efficiency as described previously (40). HCV core protein expression in trans-
fected cells was also detected by Western blot analysis using specific antibodies.
Enzyme-linked immunosorbent assay for cytokines. Levels of secreted cyto-
kines IL-12 (p70) and IL-10 in human DC culture medium were measured using
a BD OptEIA set human cytokine kit following the supplier’s protocol (BD
Biosciences, San Jose, CA). Briefly, microwells were coated with capture anti-
body and incubated overnight at 4°C. Plates were blocked and incubated with
standard, sample, and control. Detection antibody and streptavidin-horseradish
peroxidase reagents were added to each well. Finally, substrate solution was
added and the mixture was incubated for color development and stopped by the
addition of H
2
SO
4
and read in an enzyme immunoassay reader. Cytokine con
-
centrations were determined by comparison with a standard curve generated
using highly purified recombinant cytokine. All samples were assayed in dupli-
cate, and data are expressed as picograms per milliliter.
DC function. The experimental design for the experiment was similar to that
of work previously described (15, 49). For each condition, 4 10
4
DCs were
resuspended in a total volume of 200 l RPMI medium, RPMI medium plus
recombinant human IL-10 (BD Biosciences), control hepatocyte culture me-
dium, or supernatants from HCV-infected hepatocytes. Control hepatocyte cul-
ture media were included as conditioned medium from either uninfected IHH
(CM1 medium) or Huh-7.5 cells (CM2 medium), and virus-containing superna-
tants were generated by infecting these cells with HCV1 (clone H77) or HCV2
(clone JFH1), respectively, at a multiplicity of infection of approximately 0.5. The
DCs and the various conditioned media were incubated for 48 h in the presence
or absence of 10 g/ml Mycobacterium tuberculosis culture filtrate (Mycobacteria
Research Laboratories, Colorado State University, Fort Collins, CO). DCs were
then washed twice with fresh RPMI medium and irradiated before addition of
1 10
6
autologous peripheral blood mononuclear cells labeled with carboxy
-
fluoroscein succinimidyl ester (CFSE) (Vybrant CFDA-SE; Molecular Probes/
Invitrogen, Eugene, OR). Cultures (1 ml) were incubated for 7 days at 37°C and
5% CO
2
, after which the cells were restimulated for 2 h with phorbol myristate
acetate and ionomycin (Sigma Aldrich, Saint Louis, MO) and counted. Cells
were surface stained with anti-CD3 peridinin-chlorophyll-protein complex, anti-
human IFN- allophycocyanin, anti-human CD4 Alexa 700, and anti-human
CD8 Pac Blue (BD Biosciences), permeabilized and fixed for intracellular IFN-
staining, and analyzed by flow cytometry. Absolute numbers of antigen-specific
CD3
/CD4
/CFSE
lo
/IFN-
cells were calculated by multiplying the frequency
of each subset by the total number of viable cells.
RESULTS
HCV polyprotein expression inhibits MHC class II upregu-
lation in IFN--treated HT1080 cells. HT1080 cells were gen-
erated from fibrosarcoma tissue and originally subcultivated
under conditions that eliminate the growth of fibroblasts and
favor that of epithelial cells. HT1080 cells are sensitive to the
induction of HLA-DR molecules by IFNs. IFN- activates
antigen presentation by several mechanisms, including tran-
scriptional upregulation of HLA-DR molecules and proteases
involved in the immunoproteasome that are important for pro-
cessing antigenic peptide. We have examined whether the
presence of HCV proteins perturbs the upregulation of
HLA-DR molecules in IFN--treated HT1080 cells. For this,
HT1080 cells were stably transfected with plasmid DNA en-
coding a partial or HCV-FL genomic region, resulting in trans-
fected cells expressing HCV proteins (5). Mock-transfected
cells were used in parallel as negative controls. Cells were
treated with IFN- (500 U/ml) as an inducer of HLA-DR
expression and analyzed by flow cytometry. Treatment of
mock-transfected HT1080 cells with IFN- caused an upregu-
lation of HLA-DR surface expression (Fig. 1A and B). HT1080
cells expressing HCV core protein also responded to IFN-
similarly, with upregulation of HLA-DR molecules (Fig. 1C
and D). However, HT1080 cells transfected with HCV-FL
genomic region did not upregulate HLA-DR expression after
stimulation with IFN- (Fig. 1E and F). The present results
indicated that inhibition of HLA-DR expression is associated
with HCV polyprotein expression in IFN--treated HT1080
cells.
HCV protein expression downregulates CIITA, GAS, and
HLA-DR promoter activities. MHC class II expression is ex-
quisitely controlled at a cell type-specific level and is precisely
fine tuned and regulated, primarily by CIITA (41). Indeed,
CIITA is the primary factor activating the expression of the
class II genes necessary for the exogenous pathway of antigen
processing and presentation (16). CIITA is a highly regulated
non-DNA-binding coactivator that exhibits a remarkable de-
gree of specificity for MHC class II genes. CIITA expression is
regulated mainly at the level of transcription. CIITA is in-
volved in both constitutive and inducible class II gene expres-
sion. The regulation of antigen expression by CIITA is complex
and differs in various cell types, depending on the relative
activities of CIITA promoters (12). We determined whether
IFN- could act directly to induce CIITA gene transcription in
FIG. 3. Characterization of immature and mature human DCs. Monocytes were treated for 6 days with IL-4 and granulocyte-macrophage
colony-stimulating factor and then either rested in the same medium for 1 more day (immature DC) or treated with activation cocktail (IL-1, IL-6,
tumor necrosis factor alpha, and PGE2) for 1 more day (mature DC). (A) Light microscopic views (100 magnification) of two monocyte-derived
DCs are shown. (B) Granularity and surface expression results for the indicated markers (HLA-DR, Lin1, and CD80) in immature and mature
DCs are also shown.
V
OL. 82, 2008 INHIBITORY EFFECTS OF HCV ON ANTIGEN-PRESENTING CELLS 3323
Page 4
3324
Page 5
HT1080 cells. For this, the IFN--inducible promoter of the
human CIITA gene was used to drive expression of the lucif-
erase reporter gene. The CIITA-luciferase plasmid construct
was transiently transfected into HT1080 cells and induced with
IFN-, and luciferase activity levels were determined (Fig. 2A).
A very low level of basal transcriptional activity of the reporter
construct was detected in the absence of IFN-, while a five- to
sixfold increase in induction of CIITA promoter activity was
observed upon stimulation with IFN- (data not shown).
HT1080 cells expressing HCV core displayed CIITA promoter
induction similar to that seen with the vector control, while
HT1080 cells transfected with the HCV-FL construct were
found to have markedly lower levels of IFN--induced CIITA
promoter activity. An analysis of GAS (Fig. 2B) and HLA-DR
(Fig. 2C) promoters exhibited similar results, demonstrating
that HCV core protein did not have inhibitory effects, whereas
HCV-FL expression did inhibit promoter activity in IFN--
treated HT1080 cells.
HCV inhibits the maturation of dendritic cells. We focused
our studies in the above-described experiments using HT1080
cells to understand the molecular mechanisms controlling
MHC class II-inducible expression as a model system. How-
ever, HT1080 cells are not representative of APCs involved in
the initial induction of immune functions. Next, we have fo-
cused on studies of DCs, which are important for the initiation
of immune responses to foreign antigens, because of their
competence in the capture and presentation of antigen to T
cells. After antigen internalization, the DCs themselves un-
dergo a process of maturation, migration, and relocation (7).
During maturation, cell morphology alters and DCs upregulate
MHC class II and a variety of costimulatory molecules. A
typical characterization profile of immature and mature DCs
used in our studies is shown (Fig. 3). HCVcc inhibited DC
maturation even though HCV core and NS4a expression were
observed in 0.7% and 0.5% of DCs, respectively (data not
shown). DCs exposed to HCVcc were examined for cell surface
expression of HLA-DR by FACS analysis. As shown with a
representative preparation of DCs (Fig. 4A), immature DCs
expressed HLA-DR, and the level of expression increased to
some degree with MC treatment. However, supernatants from
HCV-infected hepatocytes markedly inhibited HLA-DR ex-
pression on these DCs. Despite culture conditions which
minimized the increases in HLA-DR expression with DC mat-
uration, components from the supernatants collected from
HCV-infected cells suppressed HLA-DR expression below the
levels detected at baseline in immature DCs. MC-induced
HLA-DR expression was also reduced in DCs incubated with
IL-10 (not shown). MC-induced DCs incubated with CM from
uninfected IHH displayed a trend for a lower mean fluores-
cence of HLA-DR staining compared with cells incubated with
MC alone. The percentages of HLA-DR-positive cells and
mean HLA-DR fluorescence obtained in similar experiments
using DCs from five different healthy volunteers are shown
(Fig. 4B and C). Percentages of both HLA-DR-positive cells
and mean fluorescence intensity were significantly reduced fol-
lowing incubation with HCVcc. Statistical analyses of percent-
ages of HLA-DR-positive cells and HLA-DR mean fluores-
cence gave P values of 0.05 by Wilcoxon matched-pair test in
comparisons of DCs incubated with virus (HCV1 or HCV2) to
DCs incubated with control medium (CM1 or CM2).
During maturation, DCs upregulate MHC, adhesion, and
costimulatory molecules, including CD40, CD80, and CD83.
DCs from the five different healthy volunteers (Fig. 4B and C)
were examined for MC-induced costimulatory molecule ex-
pression after incubation with supernatants from HCV2-in-
fected hepatocytes (Table 1). DCs expressed increased levels
of maturation markers CD40, CD80, and CD83 upon incuba-
tion with MC. In contrast, DCs incubated with HCVcc ex-
pressed lower MC-induced levels of these markers. Interest-
ingly, conditioned medium from mock-infected hepatocytes
also had a negative effect on these additional markers of DC
maturation but to a much lower extent. The differences in the
percentages of CD40-, CD80-, or CD83-positive cells were
consistently lower in DCs incubated with HCV, reflecting a less
mature phenotype.
Cytokine expression in DCs exposed to HCV. DCs regulate
virus-specific immune responses that are crucial for virus erad-
ication through the production of stimulatory and suppressive
cytokines. Th1 cytokines are required for host antiviral im-
mune responses, while Th2 cytokines can inhibit the develop-
ment of these effector mechanisms. IL-12 produced by DCs
can bias for the development of Th1 responses. IL-10 produc-
tion by DCs, in contrast, is associated with tolerogenic DCs
(28). The ability of HCVcc to influence cytokine secretion by
DCs has yet to be explored. We investigated the effects of
FIG. 4. Expression of HLA-DR on cell surface upon exposure of human monocyte-derived DCs to HCV. FACS analysis and a typical expression
profile of HLA-DR on the cell surface of monocyte-derived DCs from a healthy volunteer, and after stimulation with MC as a positive control are
shown. (A) DCs were treated separately with conditioned medium (CM2) from hepatocytes as a mock control or exposed to cell culture-grown
HCV2. (B and C) FACS analysis showing inhibition of HLA-DR expression on cell surface upon exposure of DCs from five healthy volunteers
to cell culture-grown HCV genotype 1a or genotype 2a. Results are presented as percentages of positive cells (B) and mean fluorescence intensity
(C). HLA-DR expression was analyzed using immature DCs, cells treated with MC, and cells treated with MC and CM from IHH (CM1) or
Huh-7.5 (CM2) and HCVcc genotype 1a (HCV1) or genotype 2a (HCV2). , P 0.05 by Wilcoxon matched-pair test in comparison with DCs
treated with MC alone.
TABLE 1. Expression of costimulatory molecules in DCs
determined by FACS analyses
a
Costimulatory
molecule
%( SE) of indicated molecule
DC DC MC
DC MC
CM2
DC MC
HCV2
CD40
b
49.3 4 64.7 5 61.1 6 50.9 8
CD80
c
19.2 5 41.4 7 37.6 8 24.5 7
CD83
b
29.1 7 45.5 11 39.3 11 24.6 9
a
Data represent results obtained with different individual donors.
b
For DC MC versus DC MC HCV2, P 0.05; for DC MC CM2
versus DC MC HCV2, P 0.05.
c
For DC MC versus DC MC HCV2, P 0.05; for DC MC CM2
versus DC MC HCV2, P 0.07.
VOL. 82, 2008 INHIBITORY EFFECTS OF HCV ON ANTIGEN-PRESENTING CELLS 3325
Page 6
HCVcc on IL-12 (p70) and IL-10 secretion by use of MC-
induced human DCs. Monocyte-derived DCs from five differ-
ent healthy volunteers were induced with IL-1, TNF, IL-6,
and PGE2 in the presence of HCVcc, and IL-10/IL-12 secre-
tion results were analyzed. DCs stimulated with the MC did
not produce IL-12 (p70) in the presence or absence of HCVcc
(data not shown). This result was probably related to the find-
ing that PGE2 inhibits IL-12 production (24). On the other
hand, a significant increase in IL-10 secretion was observed in
MC-induced DC treated with HCVcc (Fig. 5). Mock- or HCV-
infected hepatocyte medium from either IHH (CM1) or Huh-
7.5 cells (CM2) did not contain a detectable quantity of IL-10
(data not shown). Therefore, culture medium of HCV-infected
hepatocytes used as a source of HCVcc did not induce IL-12
production but instead induced IL-10 secretion from DCs.
Incubation of DCs with HCVcc impairs antigen-specific T-
cell activation. To determine the functional capacity of hu-
man DCs exposed to HCV, we used an assay to measure the
ability of DCs to stimulate proliferation of and effector
cytokine secretion in autologous T cells. DCs from purified
protein derivative-positive donors were incubated for 48 h
with M. tuberculosis culture filtrate antigens in the presence
or absence of HCV-containing hepatocyte culture medium.
By flow cytometry, we were able to determine for each
condition the number of T cells (CD4
or CD8
) that had
proliferated and that secreted IFN- upon restimulation
(Fig. 6). For both donors, the presence of HCV in the DC
supernatants effectively inhibited the proliferation and cy-
tokine secretion of mycobacterium-specific T cells. As indi-
cated, this was observed for both genotypes of HCVcc (HCV1
and HCV2). On the other hand, conditioned medium from
IHH (CM1) and Huh-7.5 cells (CM2), used to support HCV
growth, enhanced proliferation and cytokine secretion. This
could have been due to the differences in composition of cul-
ture media for two different cell types used in this study and the
presence of a number of secretory components in the condi-
tioned medium of the hepatocytes (6). Furthermore, the effect
of the use of HCV-containing hepatocyte culture medium
seemed to be broad and at least as potent as the addition of
recombinant exogenous IL-10, since both the subsets of effec-
tor T cells were inhibited by coculturing with HCV. These
results provided a clear indication that HCV inhibits the anti-
gen presentation function and T-cell-stimulatory capacity
of DCs.
FIG. 5. IL-10 secretion is increased in HCV-exposed DC cultures.
An enzyme-linked immunosorbent assay-based cytokine assay was per-
formed using culture medium from cells incubated alone or treated
with MC, conditioned medium from naı¨ve cells (CM1 or CM2), and
cell culture-grown HCV1 or HCV2. , P 0.05 by the Wilcoxon
matched-pair test in comparisons of CM- and HCV-treated DCs.
FIG. 6. DC function is impaired in the presence of HCV. Human monocyte-derived dendritic cells from purified protein derivative-
positive donors were fed mycobacterial culture filtrate antigen (Ag) in the absence or presence of HCV1, HCV2, or conditioned media from
uninfected hepatocytes (CM1 or CM2). After 48 h, the DCs were washed and incubated with CFSE-labeled autologous peripheral blood
mononuclear cells for 7 days. Subsets of CD3
lymphocytes (CD4
or CD8
) were analyzed by flow cytometry for dilution of CFSE and
intracellular staining of IFN-. Based on cell counts in the 7-day cultures, the absolute numbers of each subset that had proliferated and
secreted IFN- were calculated.
3326 SAITO ET AL. J. V
IROL.
Page 7
DISCUSSION
In this study, we investigated the regulation of IFN--induc-
ible HLA-DR antigen expression in HT1080 cells stably ex-
pressing HCV proteins as a model cell line. HT1080 cells have
been extensively used for studies of IFN- signaling (33). Our
results demonstrated that polyprotein expression from an
HCV-FL construct inhibits HLA-DR expression in IFN--
treated HT1080 cells. Transient expression of the HCV-FL
also downregulated CIITA, GAS, and HLA-DR promoter ac-
tivities in IFN--treated HT1080 cells, although we do not
know at this time the mechanisms for downregulation. CIITA
is a global regulator for the expression of genes involved in
antigen presentation (10). The presence of HCV proteins leads
to the downregulation of MHC class II antigen presentation on
the cell surface. Our results further provided direct evidence
that monocyte-derived DCs exposed to HCVcc have decreased
expression of several markers of DC maturation, secrete sig-
nificant amounts of IL-10, and are significantly impaired in
their ability to present antigen. Our observations are in agree-
ment with earlier reports suggesting that monocyte-derived
DCs from chronically HCV-infected patients do not respond
to maturation stimulus antigens and maintain their immature
phenotype (1, 3). Together, these results suggest that the neg-
ative effects of HCV on APC function could lead to reduced
immunogenicity in vivo.
A maturation defect in monocyte-derived DCs generated
from chronic HCV-infected donors has been reported earlier
(1, 3, 27). Distinct and contrasting dysfunction features of
plasmacytoid and myeloid DC subsets were also observed dur-
ing chronic HCV infection (2). Myeloid DCs derived from
persons with chronic HCV infection displayed defects in IL-12
p70 production that were related to IL-10 activity and that
could be overcome by treatment of the DCs with CD40L and
IFN- (17). However, these observations fail to explain why
HCV infection is not characterized by general immunosup-
pression. Subsequent studies of chronically HCV-infected
chimpanzees (44) or humans (32) did not suggest a defect in
DCs. A significant level of HCV protein expression or virus
replication was not observed upon incubation of DCs with
HCV. Infected hepatocytes might be secreting unknown con-
stituent(s) and while in circulation may be diluted enough not
to cause a significant generalized effect on DCs. On the other
hand, HCV has been shown to infect not only liver cells but
also extrahepatic tissues, including DCs (20, 31). Virus repli-
cation may occur in only a proportion of DCs in patients with
chronic HCV infection, allowing the uninfected DCs to nor-
mally present non-HCV antigens to T cells (45). This would
explain how patients with chronic hepatitis C exhibit a selective
deficit of anti-HCV immunity with preservation of normal im-
mune response to unrelated antigens.
Cellular immune responses are critical for the clearance of
HCV (19). Failure to mount a potent and broad T-cell repertoire
response results in persistent HCV infection. It has been sug-
gested that HCV subverts cellular immunity by inducing IL-10,
which in turn inhibits the activation of DC and development of
Th1 cells (9). This may or may not be a direct effect from the
culture medium of hepatocytes harboring HCV growth or HCV
interacting with the DC surface. IL-10 can be produced by mono-
cytes, DCs, and certain subsets of CD4
T regulatory cells. It is
possible that IL-10 induced in DCs by HCV or factors produced
by HCV-infected hepatocytes may play a role in downregulating
HCV-specific immunity in patients (14). The presence of DCs in
the liver facilitates presentation of viral antigens to both CD4
and CD8
T-cell populations. Incubation of DC with HCVcc
displayed an immunosuppressive effect upon M. tuberculosis cul-
ture filtrate with respect to the induction of CD4
and CD8
T-cell responses. Thus, our studies also provided evidence that
HCV can interfere with antigen presentation, which could be a
critical mechanism for HCV persistence. HCV has developed
multiple strategies to escape immunity and persist within the host.
A lower CD8
T-cell response induced during HCV infection
may be dependent upon reduced activation of CD4
T cells. The
importance of CD4
T cells in cytotoxic T lymphocyte activity has
been shown with chronic lymphocytic choriomeningitis virus in-
fection in mice (50). Viral persistence is facilitated by the pres-
ence of unresponsive CD8
T cells because of the absence of an
efficient CD4
response. Therefore, downregulation of APC
function associated with IL-10 production, especially by intrahe-
patic infiltrating DCs, may lead to a crippled immune system that
is incapable of responding with an appropriate immune response
against HCV. Further work should elucidate the mechanisms by
which specific HCV proteins regulate MHC class II expression
and inhibit DC maturation and the interplay of these effects for
viral persistence and pathogenesis.
ACKNOWLEDGMENTS
We thank Jenny P.-Y. Ting for providing CIITA and HLA-DR
promoters and Lin Cowick for preparation of the manuscript.
This work was supported by research grant AI068769 from the
National Institutes of Health.
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3328 SAITO ET AL. J. VIROL.
Page 9
  • Source
    • "However, cell surface expression of HLA class II molecules is not possible until the invariant chain is degraded by a protease such as cathepsin. Hepatitis C virus is known to affect dendritic cell function and maturation and has been shown to inhibit HLA class II (HLA-DR) expression on dendritic cells (Siavoshian et al., 2005; Averill et al., 2007; Saito et al., 2008). Subsequent studies have shown that dendritic cells exposed to HCV exhibit decreased expression of Cathepsin S with a corresponding decrease of HLA-DR expression on the cell surface, mainly mediated through the HCV proteins core and NS5A (Kim et al., 2012). "
    [Show abstract] [Hide abstract] ABSTRACT: Some of the central challenges for developing effective vaccines against HIV and hepatitis C virus (HCV) are similar. Both infections are caused by small, highly mutable, rapidly replicating RNA viruses with the ability to establish long-term chronic pathogenic infection in human hosts. HIV has caused 60 million infections globally and HCV 180 million and both viruses may co-existent among certain populations by virtue of common blood-borne, sexual or vertical transmission. Persistence of both pathogens is achieved by evasion of intrinsic, innate and adaptive immune defenses but with some distinct mechanisms reflecting their differences in evolutionary history, replication characteristics, cell tropism and visibility to mucosal versus systemic and hepatic immune responses. A potent and durable antibody and T cell response is a likely requirement of future HIV and HCV vaccines. Perhaps the single biggest difference between the two vaccine design challenges is that in HCV, a natural model of protective immunity can be found in those who resolve acute infection spontaneously. Such spontaneous resolvers exhibit durable and functional CD4+ and CD8+ T cell responses. However frequent re-infection suggests partial or lack of protective immunity against heterologous HCV strains, possibly indicative of the degree of genetic diversity of circulating HCV genotypes and subtypes. There is no natural model of protective immunity in HIV, however studies of “elite controllers”, or individuals who have durably suppressed levels of plasma HIV RNA without antiretroviral therapy has provided the strongest evidence for CD8+ T cell responses in controlling viremia and limiting reservoir burden in established infection. Here we compare and contrast the specific mechanisms of immune evasion used by HIV and HCV, which subvert adaptive human leucocyte antigen (HLA)-restricted T cell immunity in natural infection, and the challenges these pose for designing effective preventative
    Full-text · Article · Oct 2014 · Frontiers in Microbiology
  • Source
    • "HCV can have an inhibitory effect on antigen presenting cells, resulting in reduction of antigen-specific T-cell activation. These effects may contribute to the overall low level of immunogenicity of HCV observed in chronically infected patients (Saito et al., 2008 ). HCV has an inhibitory role on cathepsin S-mediated major histocompatibility complex (MHC) class II maturation, which may contribute to weak immunogenicity of viral antigens in chronically infected humans (Kim et al., 2012). "
    [Show abstract] [Hide abstract] ABSTRACT: Hepatitis C virus (HCV) often causes persistent infection, and is an important factor in the etiology of fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). There are no preventive or therapeutic vaccines available against HCV. Treatment strategies of HCV infection are likely to improve with recently discovered direct antiviral agents (DAAs). However, a proportion of patients still progress to liver failure and/or HCC despite having been cured of the infection. Thus, there is a need for early diagnosis and therapeutic modalities for HCV related end stage liver disease prevention. HCV genome does not integrate into its host genome, and has a predominantly cytoplasmic life cycle. Therefore, HCV mediated liver disease progression appears to involve indirect mechanisms from persistent infection of hepatocytes. Studying the underlying mechanisms of HCV mediated evasion of immune responses and liver disease progression is challenging due to the lack of a naturally susceptible small animal model. We and other investigators have used a number of experimental systems to investigate the mechanisms for establishment of chronic HCV infection and liver disease progression. HCV infection modulates immune systems. Further, HCV infection of primary human hepatocytes promotes growth, induces phenotypic changes, modulates epithelial mesenchymal transition (EMT) related genes, and generates tumor initiating stem-like cells (TISCs). HCV infection also modulates microRNAs (miRNAs), and influences growth by overriding normal death progression of primary human hepatocytes for disease pathogenesis. Understanding these ob-servations at the molecular level should aid in developing strategies for additional effective therapies against HCV mediated liver disease progression.
    Preview · Article · Aug 2014 · EXCLI Journal
  • Source
    • "HCV can have an inhibitory effect on antigen presenting cells, resulting in reduction of antigen-specific T-cell activation. These effects may contribute to the overall low level of immunogenicity of HCV observed in chronically infected patients (Saito et al., 2008 ). HCV has an inhibitory role on cathepsin S-mediated major histocompatibility complex (MHC) class II maturation, which may contribute to weak immunogenicity of viral antigens in chronically infected humans (Kim et al., 2012). "
    [Show abstract] [Hide abstract] ABSTRACT: Hepatitis C virus (HCV) often causes persistent infection, and is an important factor in the etiology of fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). There are no preventive or therapeutic vaccines available against HCV. Treatment strategies of HCV infection are likely to improve with recently discovered direct antiviral agents (DAAs). However, a proportion of patients still progress to liver failure and/or HCC despite having been cured of the infection. Thus, there is a need for early diagnosis and therapeutic modalities for HCV relatedend stage liver disease prevention. HCV genome does not integrate into its host genome, and has a predominantly cytoplasmic life cycle. Therefore, HCV mediated liver disease progression appears to involve indirect mechanisms from persistent infection of hepatocytes. Studying the underlying mechanisms of HCV mediated evasion of immune responses and liver disease progression is challenging due to the lack of a naturally susceptible small animal model. We and other investigators have used a number of experimental systems to investigate the mechanisms for establishment of chronic HCV infection and liver disease progression. HCV infection modulates immune systems. Further, HCV infection of primary human hepatocytes promotes growth, induces phenotypic changes, modulates epithelial mesenchymal transition (EMT) related genes, and generates tumor initiating stem-like cells (TISCs). HCV infection also modulates microRNAs (miRNAs), and influences growth by overriding normal death progression of primary human hepatocytes for disease pathogenesis. Understanding these observations at the molecular level should aid in developing strategies for additional effective therapies against HCV mediated liver disease progression.
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