Morphine Withdrawal Enhances Hepatitis C Virus Replicon Expression

Article (PDF Available)inAmerican Journal Of Pathology 167(5):1333-40 · December 2005with22 Reads
DOI: 10.1016/S0002-9440(10)61220-5 · Source: PubMed
Abstract
We previously demonstrated that morphine enhances hepatitis C virus (HCV) replication in human hepatic cells. Here we describe the impact of morphine withdrawal (MW), a recurrent event during the course of opioid abuse, on HCV replicon expression in human hepatic cells. MW enhanced both viral RNA and protein expression in HCV replicon cells. Blocking opioid receptors by treatment with naloxone after morphine cessation (precipitated withdrawal, PW) induced greater HCV replicon expression than MW. Investigation of the mechanism responsible for MW- or PW-mediated HCV enhancement showed that both MW and PW inhibited the expression of endogenous interferon-alpha (IFN-alpha) in the hepatic cells. This down-regulation of intracellular IFN-alpha expression was due to the negative impact of MW or PW on IFN-alpha promoter activation and on the expression of IFN regulatory factor 7 (IRF-7), a strong transactivator of the IFN-alpha promoter. In addition, both MW and PW inhibited the anti-HCV ability of recombinant IFN-alpha in the hepatic cells. These in vitro observations support the concept that opioid abuse favors HCV persistence in hepatic cells by suppressing IFN-alpha-mediated intracellular innate immunity and contributes to the development of chronic HCV infection.

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Available from: David S Metzger
Immunopathology and Infectious Diseases
Morphine Withdrawal Enhances Hepatitis C Virus
Replicon Expression
Chuan-Qing Wang,*
Yuan Li,*
Steven D. Douglas,* Xu Wang,*
David S. Metzger,
Ting Zhang,* and
Wen-Zhe Ho*
From the Department of Pediatrics,*Division of Allergy and
Immunology, Joseph Stokes, Jr., Research Institute at The
Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania;
the Department of Psychiatry,
The Center for Studies of
Addiction, University of Pennsylvania School of Medicine,
Philadelphia, Pennsylvania; and the Division of Clinical
Laboratory,
The Children’s Hospital of Fudan University,
Shanghai, China
We previously demonstrated that morphine enhances
hepatitis C virus (HCV) replication in human hepatic
cells. Here we describe the impact of morphine with-
drawal (MW), a recurrent event during the course of
opioid abuse, on HCV replicon expression in human
hepatic cells. MW enhanced both viral RNA and
protein expression in HCV replicon cells. Blocking
opioid receptors by treatment with naloxone after
morphine cessation (precipitated withdrawal, PW)
induced greater HCV replicon expression than MW.
Investigation of the mechanism responsible for MW-
or PW-mediated HCV enhancement showed that both
MW and PW inhibited the expression of endogenous
interferon-
(IFN-
) in the hepatic cells. This down-
regulation of intracellular IFN-
expression was due
to the negative impact of MW or PW on IFN-
pro-
moter activation and on the expression of IFN regu-
latory factor 7 (IRF-7), a strong transactivator of the
IFN-
promoter. In addition, both MW and PW inhib-
ited the anti-HCV ability of recombinant IFN-
in the
hepatic cells. These in vitro observations support the
concept that opioid abuse favors HCV persistence in
hepatic cells by suppressing IFN-
-mediated intracel-
lular innate immunity and contributes to the develop-
ment of chronic HCV infection. (Am J Pathol 2005,
167:1333–1340)
Injection drug users (IDUs) are the largest group at risk
for hepatitis C virus (HCV) infection in developed coun-
tries.
1–6
HCV infection rate among IDUs is extremely high
generally ranging from 70 to more than 90% in the United
States.
2,4
IDUs frequently involve the abuse of heroin. In
fact, heroin addicts are stereotypic intravenous drug
abusers. Of heroin abusers entering treatment programs,
96% reported injection as the primary route of adminis-
tration.
7
IDUs have a greater incidence of infections than
nonabusers.
8,9
IDUs have a markedly increased preva-
lence of viral hepatitis A, B, and C.
7
Although injection
drug use contributes significantly to viral infections, it is
difficult to determine whether the cause of the increased
exposure to infectious agents is due to transmission
through contaminated needles, or whether contact with
the drugs themselves, through suppression of immune
function in the host, results in greater susceptibility to
viruses.
10
In the case of HCV infection, there is little
information about whether drug abuse, such as heroin,
enhances HCV replication and promotes HCV disease
progression. This lack of knowledge about the impact of
opioid abuse on HCV disease is a major barrier to fun-
damental understanding of HCV-related morbidity and
mortality among IDUs and to the development of new
therapeutic approaches for HCV infection.
Because of the HCV epidemic, interest in studying the
impact of drug abuse, especially opiates, on HCV infec-
tion has increased greatly. This issue is now even more
important because of the association of HCV infection
with intravenous drug abuse. It is possible that the impact
of opioids on HCV infection is related to the ability of
opioids to modulate both innate and acquired immune
response. For instance, opioids have been shown to
inhibit the expression of anti-viral cytokines, such as in-
terferon-
(IFN-
)
11–14
and IFN-
.
14–16
IFN-
is one of
critical elements in the innate host defense mechanism
against HCV infection,
17,18
whereas interferon regulatory
factor 7 (IRF-7) has a critical role in rapid induction of
IFN-
expression, a central event in establishing the in-
Supported by the National Institutes of Health (grants DA-12815 and
DA-16022 to W.-Z.H. and AA-13547 to S.D.D.).
Accepted for publication July 25, 2005.
Address reprint requests to Dr. Wen-Zhe Ho, Division of Allergy and
Immunology, The Children’s Hospital of Philadelphia, University of Penn-
sylvania School of Medicine, 34th St. and Civic Center Blvd., Philadelphia,
PA 19104. E-mail: ho@email.chop.edu.
American Journal of Pathology, Vol. 167, No. 5, November 2005
Copyright © American Society for Investigative Pathology
1333
nate anti-viral responses.
18–20
IFN-
acts directly through
its anti-viral mechanism(s) or through its regulatory effect
on innate and adaptive immune responses. IFN-
is the
only anti-viral cytokine approved for clinical treatment of
chronic HCV infection. IFN-
inhibits HCV RNA expres-
sion in the replicon cells.
21,22
Our earlier work demon-
strated that morphine enhanced HCV replication in the
hepatic cells and inhibited the anti-HCV effect of IFN-
.
23
This earlier study represents an initial step in understand-
ing the interaction between opioids and HCV in the he-
patic cells. In the present study, we investigated the
impact of morphine withdrawal (MW), a crucial and re-
current event during the course of opioid abuse, on HCV
replicon expression and the anti-HCV effect of IFN-
in
human hepatic cells. We also examined the mechanism
underlying the impact of MW on HCV replicon
expression.
Materials and Methods
Cell Lines
The HCV replicon cell line (Huh.8) was obtained from Dr.
Charles Rice (The Rockefeller University and Apath,
L.L.C., St. Louis, MO). The FCA-1 cell line was a gift from
Dr. Christoph Seeger (Fox Chase Cancer Center, Phila-
delphia, PA).
22
Huh7, a human hepatoma cell line, is the
parental cell line of the HCV replicon cell lines (Huh.8 and
FCA-1). These cells were maintained in Dulbecco’s mod-
ified Eagle’s medium (Life Technologies, Inc., Grand Is-
land, NY) supplemented with 10% fetal bovine serum
(HyClone, Logan, UT), in the presence of penicillin/strep-
tomycin, L-glutamine, and minimal essential medium non-
essential amino acids. The culture media for the HCV
replicon cells contained G418 (1 mg/ml; Invitrogen Corp.,
Grand Island, NY). The cells were passaged every 3 days
and seeded in 24-well plates (10
5
cells/well). For all ex-
periments, endotoxin-free media and reagents were
used.
Reagents
Morphine sulfate (15 mg/ml) was purchased from Elkins-
Sinn, Inc. (Cherry Hill, NJ). Naloxone and rabbit poly-
clonal antibody against actin were obtained from Sigma-
Aldrich (St. Louis, MO). Recombinant human IFN-
and
rabbit polyclonal antibody to IRF-7 were obtained from
R&D Systems Inc. (Minneapolis, MN). Mouse monoclonal
antibody against HCV NS5A was obtained from ViroStat
(Portland, ME). Horseradish peroxidase-conjugated goat
anti-mouse antibody and goat anti-rabbit IgG were pur-
chased from Jackson ImmunoResearch Laboratories
(West Grove, PA).
Morphine Withdrawal
Huh7 and the HCV replicon cells (Huh.8 and FCA-1) were
plated in 24-well plates and treated with or without mor-
phine (10
6
mol/L) every 24 hours for 4 days. Morphine
was then removed by washing the cells three times with
1phosphate-buffered saline (PBS) at day 5. For pre-
cipitated withdrawal (PW) experiments, naloxone (10
8
mol/L) was added to the cell cultures immediately after
morphine was removed. The selection of concentrations
of morphine (10
6
mol/L) and naloxone (10
8
mol/L) for
this study was based on our pilot experiments and pub-
lished studies.
15,23,24
These concentrations are compa-
rable to reported therapeutic serum concentrations of
morphine and naloxone in patients.
25
There was no cy-
totoxic effect of morphine treatment on the hepatic cells,
as determined by trypan blue dye staining. To determine
whether MW or PW interferes with the anti-HCV effect of
IFN-
, Huh.8 and FCA-1 cells were incubated with IFN-
(10 U/ml) immediately after morphine was removed. The
levels of HCV and IFN-
were analyzed at 24 hours, 48
hours, and 72 hours after MW or PW.
Enzyme-Linked Immunosorbent Assay for IFN-
Enzyme-linked immunosorbent assay kits for IFN-
were
purchased from PBL Biomedical Laboratories (Piscat-
away, NJ) and the assay was performed according to the
protocol provided by the manufacturer.
Polymerase Chain Reaction (PCR) and Real-
Time Reverse Transcriptase (RT)-PCR Analyses
Total RNA was extracted from Huh7 and the HCV repli-
con cells using Tri-Reagent (Molecular Research Center,
Cincinnati, OH) as described.
26
Each of the PCR ampli-
fications included heat activation of AmpliTaq Gold for 9
minutes at 95°C, followed by 40 cycles of 95°C for 15
seconds, 52°C for 20 seconds, and 72°C for 40 seconds,
and further elongation at 72°C for 7 minutes. The specific
oligonucleotide primers used were as follows: IFN-
:5-
TTT CTC CTG CCT GAA GGA CAG-3(sense) and 5-
GCT CAT GAT TTC TGC TCT GAC A-3(anti-sense);
IRF-7: 5-CTG TGG TGG TGG GAC AGC TGC-3(sense)
and 5-CCC CAC GCG TGC TGT TCG GAG-3(anti-
sense). The oligonucleotide primers were synthesized by
Integrated DNA Technologies Inc. (Coralville, IA). HCV
real-time RT-PCR assay that we have developed was
used for the quantification of HCV RNA.
27
The real-time
RT-PCR for IFN-
mRNA was performed with the Brilliant
SYBR Green QPCP Master Mix (Stratagene, La Jolla,
CA).
Transfection and Luciferase Assays
Two plasmids (pIFNA4-Luc and wild-type pIRF-7) were
obtained from Dr. Rong-Tuan Lin
28
(McGill University,
Montreal, Quebec, Canada). The cells undergoing MW or
PW were seeded into a 24-well culture plate at the density
of 10
5
cells/well. Transfection was performed with Fu-
GENE 6 transfection reagent (Roche Molecular Bio-
chemicals, Indianapolis, IN) at a ratio of FuGENE 6:plas-
mid 6:1 (
l:
g) immediately after MW or PW. The cells in
each well were harvested 48 hours after transfection and
washed twice with 1PBS by centrifugation at 3300 g
1334 Wang et al
AJP November 2005, Vol. 167, No. 5
for 3 minutes at room temperature. The cell pellets were
lysed with reporter lysis buffer (Promega, Madison, WI).
Cell-free lysates were obtained by centrifugation at
10,000 gfor 1 minute at room temperature.
Western Blot
Total cell lysates from the replicon cells were prepared
using the lysis buffer (Promega). Protein concentrations
were determined by DC protein assay kit (Bio-Rad, Her-
cules, CA). Proteins were resuspended in NuPAGE LDS
sample buffer (Novex, San Diego, CA), heated for 5
minutes at 95°C, and then equal amounts of protein for
each sample were separated in a 10% Bis-Tris Gel in a
NuPAGE running buffer with 0.25% running buffer anti-
oxidant for 50 minutes at 200 V. Proteins were transferred
to the nitrocellulose membranes (Bio-Rad) in NuPAGE
transfer buffer at 100 V for 1 hour. The membranes were
blocked by 5% fat-free milk in PBST (0.05% Tween 20 in
PBS) for 1 hour and incubated with mouse monoclonal
antibody against NS5A (1:100) and rabbit polyclonal
antibody against actin (1:3000) diluted with 2% fat-free
milk in PBST for 1 hour at room temperature. For IRF-7
protein detection, rabbit polyclonal antibody to IRF-7 was
diluted 1:200. After washing four times for 5 minutes each
with PBST, the membrane was incubated with horserad-
ish peroxidase-conjugated goat anti-mouse antibody
(1:5000) and goat anti-rabbit IgG (1:10,000) in 2% fat-
free milk in PBST for 1 hour at room temperature and
washed four times as described above. The bound anti-
bodies were developed using SuperSignal West Pico
chemiluminescent substrate (Pierce, Rockford, IL) ac-
cording to the manufacturer’s instruction. Prestained mo-
lecular markers (Bio-Rad) were used to determine mo-
lecular weight of immunoreactive bands.
Statistical Analysis
Where appropriate, data were expressed as mean SD.
For comparison of the mean of the two groups (morphine
or MW or PW versus untreated control cells), statistical
significance was assessed by Student’s t-test. Calcula-
tions were performed with the use of Stata Statistical
Software (StataCorp., College Station, TX). Statistical sig-
nificance was defined as P0.05.
Results
MW Enhances HCV Replicon Expression
We first examined whether MW or PW enhances HCV
RNA expression in Huh.8 and FCA-1 cells. In comparison
with the untreated cells, the cells undergoing MW
showed increased levels of HCV RNA (Figure 1). Block-
ing opioid receptors by naloxone after the removal of
morphine (PW) also induced higher HCV replicon expres-
sion than untreated cells (Figure 1). A significant increase
in HCV RNA expression was observed at 48 hours after
MW or PW (Figure 1). This MW- or PW-induced increase
of HCV RNA was observed in both Huh.8 and FCA-1 cells
(Figure 1). We also investigated whether MW or PW en-
hances the expression of HCV NS5A protein, a critical
factor for HCV infection and replication.
21
The increased
Figure 1. Effect of MW or PW on HCV replicon expression. Huh.8 (A) and
FCA-1 (B) cells were incubated with or without morphine (10
6
mol/L) for
4 days, and morphine was removed at day 5. For PW, naloxone (10
8
mol/L)
was added to the cell cultures immediately after morphine was removed.
Total RNA extracted from the cell cultures was subjected to real-time RT-PCR
for HCV RNA quantification at indicated time points after MW or PW. The
data are expressed as HCV RNA levels in the cells undergoing MW or PW
relative (fold) to those in the untreated control cells (CTL), which is defined
as 1. The results shown are the mean SD of triplicate cultures, represen-
tative of three independent experiments (*P0.05, **P0.01; MW or PW
versus control).
Figure 2. Effect of MW or PW on HCV NS5A protein expression. Huh.8 and
FCA-1 cells were incubated with or without morphine (10
6
mol/L) for 4
days, and morphine was removed from the cell cultures at day 5. For PW,
naloxone (10
8
mol/L) was added to the cell cultures immediately after
morphine was removed. Total proteins extracted from the cells at 48 hours
after MW or PW were applied onto a nitrocellulose membrane and subjected
to Western blot assay using the antibodies specific to NS5A and actin. The
insets below the panels show the signal intensities of protein bands of the
representative blot expressed as densitometry scanning units (DSUs). One
representative experiment is shown. CTL, untreated control cells.
Morphine Withdrawal Enhances HCV Replicon 1335
AJP November 2005, Vol. 167, No. 5
levels of HCV NS5A protein were observed in Huh.8 and
FCA-1 cells at 48 hours after MW or PW (Figure 2).
MW Suppresses IFN-
Expression
Our earlier study showed that human hepatic cells ex-
press endogenous IFN-
that powerfully inhibits HCV
replicon expression.
29
Thus, we hypothesized that MW or
PW suppresses endogenous IFN-
expression in the he-
patic cells, through which it induces HCV replicon ex-
pression. A significant decrease in IFN-
expression in
the replicon cells (Huh.8 and FCA-1) was observed at 48
hours after MW or PW (Figure 3). Because there is a
reciprocal interaction between intracellular IFN-
and
HCV,
29
we also examined whether MW or PW alters en-
dogenous IFN-
expression in Huh7 cells, the parent cell
line for the replicon cells. Huh7 cells undergoing MW or
PW showed lower levels of IFN-
mRNA than control
Huh7 cells (Figure 4A). A significant decrease in endog-
enous IFN-
expression in Huh7 cells was observed at 48
hours after MW or PW (Figure 4A), which is similar to the
replicon cells. We next examined whether IFN-
protein
expression is also inhibited in the hepatic cells under-
going MW or PW. Both Huh7 and the replicon cells un-
dergoing MW or PW had decreased levels of IFN-
pro-
tein in comparison to the control cells (Figure 4B).
MW Inhibits IRF-7-Induced IFN-
Promoter
Activity and IRF-7 Expression
To investigate the mechanism(s) responsible for MW- or
PW-mediated down-regulation of endogenous IFN-
ex-
pression, we examined whether MW or PW suppresses
IFN-
promoter activity in the hepatic cells. Because
IRF-7 is a strong activator of IFN-
promoter, we used
IRF-7 to activate IFN-
promoter. IRF-7, when introduced
into the hepatic cells, significantly activated IFN-
pro-
moter. However, this IRF-7-induced IFN-
promoter ac-
tivity was significantly suppressed in the hepatic cells
Figure 3. The time point action of MW or PW on endogenous IFN-
expression in the replicon cells. Huh.8 (A) and FCA-1 (B) cells were pro-
cessed under the identical conditions as described in Figure 1. Total RNA
extracted from the cell cultures was subjected to the real-time RT-PCR for
IFN-
mRNA quantitation at indicated time points after MW or PW. The data
are expressed as IFN-
RNA levels in the cells undergoing MW or PW relative
(%) to those in the control cells (CTL), which is defined as 100. The results
shown are the mean SD of triplicate cultures, representative of three
independent experiments (*P0.05, **P0.01; MW or PW versus control).
Figure 4. Effect of MW or PW on endogenous IFN-
expression in the
hepatic cells. Huh7, Huh.8, and FCA-1 cells were incubated with or without
morphine (10
6
mol/L) for 4 days, and morphine was then removed at day
5. For PW, naloxone (10
8
mol/L) was added to the cultures immediately
after morphine was removed. A: RNA extracted from the cells was subjected
to real-time RT-PCR for IFN-
RNA quantification at 48 hours after MW or PW.
The data are expressed as IFN-
RNA levels in the cells undergoing MW or
PW relative (%) to those in the untreated control cells ( CTL), which is defined
as 100. B: Supernatants were collected from the cell cultures under identical
conditions described in Afor IFN-
protein quantification by enzyme-linked
immunosorbent assay. The results shown in both Aand Bare the mean SD
of triplicate cultures, representative of three independent experiments (*P
0.05, **P0.01; MW or PW versus control).
1336 Wang et al
AJP November 2005, Vol. 167, No. 5
undergoing MW or PW (Figure 5). The levels of IFN-
promoter suppression in the HCV replicon cells (Huh.8
and FCA-1) undergoing MW or PW were higher than
those in Huh7 cells (Figure 5). Both MW and PW inhibited
IRF-7 expression at both transcriptional (Figure 6A) and
translational (Figure 6B) levels.
MW Compromises the Anti-HCV Effect of IFN-
Recombinant IFN-
remains the treatment of choice for
chronic HCV infection today. IFN-
treatment, however, is
effective in only 30 to 50% of treated patients.
30–32
Be-
cause IDUs are the largest group at risk for HCV infec-
tion, it is critical to understand whether MW plays a role in
resistance to IFN-
therapy. We examined whether MW
or PW has a negative impact on the anti-HCV effect of
recombinant IFN-
in the replicon cells. IFN-
, when
added to Huh.8 and FCA-1 cell cultures, significantly
inhibited HCV replicon expression (Figure 7). This anti-
HCV ability of IFN-
, however, was significantly dimin-
ished (from 85% to 60% and to 50% for MW and
PW, respectively) in the replicon cells (both Huh.8 and
FCA-1 cells) undergoing MW or PW (Figure 7).
Discussion
Heroin addicts have an increased susceptibility to a va-
riety of infectious diseases.
9
Heroin addicts are a high-
risk group for HCV infection and the development of
chronic HCV disease.
33–38
In our earlier studies, we
showed that morphine, the active metabolite of heroin,
enhances HCV replication in the hepatic cells.
23
The
present study focused on the impact of MW on HCV
replication and on the anti-HCV effect of IFN-
. Physical
dependence on morphine is characterized by the occur-
rence of an abstinence or withdrawal syndrome on ter-
mination of the drug. Abstinence syndromes occur after
either abrupt withdrawal (cessation of the drug) or PW
(with or without drug cessation in conjunction with the
administration of an opioid antagonist). Naloxone-precip-
itated opioid withdrawal is a validated model for testing
medications to treat opiate withdrawal.
39
In our study,
Huh7 and the HCV replicon cells were pretreated with
morphine for 4 days to induce a tolerant/dependent state,
followed by complete morphine removal or PW (addition
of naloxone to the cell cultures immediately after removal
of morphine). Using this cell model, we have for the first
time demonstrated that MW or PW enhanced HCV repli-
con expression in the hepatic cell model. When opioid
receptors are blocked by naloxone after removal of mor-
phine, there is a greater HCV replicon expression than
MW. It is possible that the removal of exogenous mor-
phine results in endogenous opioid expression in the
hepatic cells, which may reduce the impact of MW,
Figure 5. Effect of MW or PW on IFN-
promoter activity. Huh7, Huh.8, and
FCA-1 cells were incubated with or without morphine (10
6
mol/L) for 4
days, and morphine was removed at day 5. For PW, naloxone (10
8
mol/L)
was added to the cell cultures immediately after morphine was removed. The
cells were then co-transfected with the IRF-7 plasmid and the IFN-
promoter
plasmid (IFNA4) linked to a luciferase gene. IRF-7-induced activation of
IFN-
promoter was determined by luciferase activity at 48 hours after
transfection. The data are expressed as IFN-
promoter activity in the cells
undergoing MW or PW relative (%) to those in the untreated control cells
(CTL), which is defined as 100. The results shown are the mean SD of
triplicate cultures, representative of three independent experiments (*P
0.05, **P0.01; MW or PW versus control).
Figure 6. Effect of MW or PW on IRF-7 expression. Huh7 and the replicon
cells were incubated with or without morphine (10
6
mol/L) for 4 days, and
morphine was removed at day 5. For PW, naloxone (10
8
mol/L) was added
to the cultures immediately after morphine was removed. A: RNA extracted
from the hepatic cells was subjected to conventional RT-PCR for IRF-7 RNA
and
-actin RNA at 48 hours after MW or PW. The data in Aare expressed as
IRF-7 RNA level in the cells undergoing MW or PW relative (%) to those in the
untreated control cells (CTL), which is defined as 100. The results shown are
the mean SD of triplicate cultures, representative of three independent
experiments (*P0.05, **P0.01; MW or PW versus control). B: Total
protein extracted from the replicon cells were applied onto a nitrocellulose
membrane and subjected to Western blot assay at 48 hours after MW or PW.
The insets below the panels in Bshow the signal intensities of protein
bands. One representative data of three experiments are shown.
Morphine Withdrawal Enhances HCV Replicon 1337
AJP November 2005, Vol. 167, No. 5
whereas addition of naloxone should precipitate effect of
MW, because naloxone blocks the binding of endoge-
nous opioids to their receptors. The MW- or PW-mediated
enhancing effect on HCV is not due to the subsequent
effect of the 4-day treatment with morphine, because
there was no significant increase in HCV RNA expression
at 24 hours after MW or PW (Figure 1). In addition, treat-
ment of the replicon cells with morphine for 6 days had no
effect on HCV replicon expression (data not shown). The
enhancing effect of MW or PW on HCV replicon expres-
sion was also demonstrated at the protein level. Both MW
and PW enhanced HCV NS5A protein expression in the
replicon cells. We selected HCV NS5A as a primary
target protein because NS5A has a critical role in HCV
infection and replication.
21
Collectively, these new obser-
vations in conjunction with our earlier findings
23
support
the notion that opioid abuse is a co-factor that promotes
HCV replication.
The mechanism(s) underlying the positive impact of
MW or PW on HCV replicon expression remains primarily
unknown. Abrupt withdrawal or PW from morphine in-
duces immunosuppression.
40,41
Morphine significantly
inhibited both IFN-
-, -
-, and -
-mediated natural anti-
viral defense pathways in human immune cells.
14–16
Mor-
phine potentiates HIV infection in vitro by interfering with
2,5-oligoadenylate synthetase pathway of IFN-
-medi-
ated anti-viral defense.
14
We showed that the intracellular
IFN-
is a potent anti-HCV cytokine in the replicon sys-
tem.
29
Although we do not know how the hepatic cells
undergo withdrawal at this point, we showed that MW and
PW significantly inhibited endogenous IFN-
expression
by the hepatic cells. It is well known that IFN-
plays an
important role not only in the innate host cell immunity but
also in regulation of cell functions. This MW- or PW-
mediated cytokine alteration results in a compromised
intracellular immunity, which contributes to enhanced
HCV replicon expression. We observed an association
between MW- or PW-mediated HCV replicon induction
and endogenous IFN-
inhibition in the replicon cells.
The maximal inhibition of endogenous IFN-
expression
in the replicon cells occurred at 48 hours after MW or PW
(Figure 3), which matched the time point when the high-
est enhancing effect of MW or PW on HCV was observed
(Figure 1). This inhibition of endogenous IFN-
expres-
sion was not due to MW- or PW-mediated enhancement
of HCV replicon expression, because MW or PW also
inhibited endogenous IFN-
expression in Huh7 cells, the
parental cell line for both Huh.8 and FCA-1. These find-
ings suggest that the inhibition of intracellular IFN-
is
responsible, at least in part, for both MW- and PW-medi-
ated induction of HCV replicon replication. Our further
investigation demonstrated that the down-regulation of
endogenous IFN-
expression was related to the inhibi-
tory effect of MW or PW on IFN-
promoter activation
(Figure 5). IRF-7 is a strong transactivator of IFN-
pro-
moter and has the ability to amplify IFN gene expres-
sion.
42–44
Our data, which demonstrate that MW or PW
not only suppressed IRF-7-mediated IFN-
promoter ac-
tivation (Figure 5) but also inhibited IRF-7 expression at
both mRNA and protein levels (Figure 6), suggest a pos-
sible molecular mechanism whereby MW or PW inhibited
endogenous IFN-
expression in the hepatic cells.
Clinical trials indicate that there is a therapeutic benefit
of IFN-
treatment in chronic HCV infection.
45,46
IFN-
-
based treatment, however, is effective in less than 50% of
treated patients.
47–49
Thus, it is of importance to deter-
mine whether morphine is an important co-factor respon-
sible for the failure of IFN-
treatment. Our earlier study
23
showed that morphine treatment of HCV replicon cells
compromised the anti-HCV effect of IFN-
. The current
study demonstrated that both MW and PW also inhibited
exogenous IFN-
-mediated anti-HCV activity in the rep-
licon cells. Thus, our findings provide a plausible inter-
pretation of the high failure rate of IFN-
therapy in IDUs.
The identification of mechanism(s) involved in morphine’s
action on the anti-HCV effect of IFN-
has the potential to
improve IFN-
-based treatment for HCV-infected IDUs.
Because the mechanism(s) responsible for the IFN-
-
mediated therapeutic effect is unknown,
22
it is not yet
possible for us to determine the mechanism(s) involved in
the action of MW or PW on anti-HCV activity of IFN-
in
this study. Thus, further studies are critical to determine
the anti-HCV mechanism(s) of IFN-
treatment and
whether MW or PW interferes with the mechanism(s).
Taken together, our data showing that both MW and
PW enhanced HCV replicon expression by suppressing
endogenous IFN-
expression in the hepatic cells may
have a significant in vivo implication because IFN-
is a
critical element in host innate immunity against viral in-
fections.
50–53
The reduction of intracellular IFN-
expres-
sion by opioids provides a favorable environment for HCV
growth in the hepatic cells. The results suggest that opi-
oid abusers experiencing periods of drug taking followed
by periods of withdrawal may be immunocompromised in
the liver. Thus, our findings that MW or PW inhibits IFN-
Figure 7. Effect of MW or PW on the anti-HCV activity of IFN-
. Huh.8 and
FCA-1 cells were incubated with or without morphine (10
6
mol/L) for 4
days, and morphine was then removed at day 5. For PW, naloxone (10
8
mol/L) was added to the cell cultures immediately after morphine was
removed. IFN-
(10 U/ml) was added into the cell cultures 1 hour after MW
or PW. RNA extracted from the cells was subjected to real-time RT-PCR for
HCV RNA and GAPDH RNA quantification at 48 hours after MW or PW. The
data are expressed as HCV RNA in the replicon cells that are undergoing MW
or PW relative (%) to the untreated control cells, which is defined as 100. The
results shown are the mean SD of triplicate cultures, representative of three
independent experiments (*P0.05, **P0.01; IFN-
-treated HCV replicon
cells undergoing MW or PW versus IFN-
-treated cells only).
1338 Wang et al
AJP November 2005, Vol. 167, No. 5
-mediated intracellular innate immunity in the hepatic
cells suggest that opioid abuse may contribute to the
chronicity of HCV infection and promote HCV disease
progression.
Acknowledgments
We thank Dr. Charles Rice (The Rockefeller University,
New York, NY) for generously providing the Huh7 and
Huh.8 cell lines, Dr. Christoph Seeger (Fox Chase Cancer
Center, Philadelphia, PA) for providing the FCA-1 cell
line, and Stephen Jasionowski for his excellent editorial
work on the manuscript.
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    • "Wang et al. reported that morphine withdrawal, a recurrent event during the course of opioid abuse, inhibits expression of endogenous IFNα and enhances HCV replicon expression in hepatic cells (Wang C.-Q. et al., 2005). Morphine inhibits IFN-α by direct interaction with IFN-α promoter and interferon regulatory factors (IRF)-5, IRF-7, and p38 (Li et al., 2003bLi et al., , 2007 Wang C.-Q. et al., 2005). These important in vitro findings were confirmed by Ye et al., who demonstrated that administration of opioids results in impairment of CD56 + T cell-mediated innate immune function (secretion of IFN-γ) through up-regulation of cytokine signaling 3 and protein inhibition of activated STAT 3 (SOCS-3), which may contribute to HCV infection and persistence in the liver (Ye et al., 2010). "
    [Show abstract] [Hide abstract] ABSTRACT: Opioids and their receptors have received remarkable attention because they have the ability to alter immune function, which affects disease progression. In vitro and in vivo findings as well as observations in humans indicate that opioids and their receptors positively or negatively affect viral replication and virus-mediated pathology. The present study reviews recent insights in the role of opioids and their receptors in viral infections and discusses possible therapeutic opportunities. This review supports the emerging concept that opioids and their receptors have both favorable and unfavorable effects on viral disease, depending on the type of virus. Targeting of the opioid system is a potential option for developing effective therapies; however caution is required in relation to the beneficial functions of opioid systems.
    Full-text · Article · Jun 2016
    • "In animals models, heroin and its metabolite, morphine, negatively affect innate immunity [8][9][10][11], and antibody production [12]. In addition, opioid administration increases the susceptibility and severity of bacterial and viral infections [13][14][15]. Studies in humans also support the role of opioids in the regulation of both innate and acquired immunity [5,[15][16][17][18][19], it is unclear, however, whether all opioids share the same immunosuppressive Black or African American 0 1 (9) 6 (30) 1 (5) Asian 0 1 (9) 4 (20) 1 (5) All other races 0 1 (9) 1 (5) 1 (5) Ethnicity 0.99 0.96 Non-Hispanic/Latino 9 (90) 10 (91) 18 (90) 19 (90) Hispanic/Latino 1 (10) 1 (9) 2 (10) 2 (properties. "
    [Show abstract] [Hide abstract] ABSTRACT: Background: Influenza vaccination is recommended for vulnerable individuals, including active drug users, to prevent influenza complications and decrease influenza spread. Recent studies suggest that opioids negatively regulate immune responses in experimental models, but the extent to which opioid use will affect the humoral responses to influenza vaccine in humans is unknown. This information is critical in maximizing vaccination efforts. Objective: To determine whether there is a difference in antibody response after influenza vaccination in heroin or methadone users compared to control subjects. Methods: We studied active heroin users, subjects on methadone maintenance treatment (MMT) and subjects that did not use any drugs before and 1 and 4 weeks after vaccination with trivalent influenza vaccine (TIV). We measured hemagglutination inhibition and microneutralization titers, and we compared geometric mean titers (GMT), and rates of seroprotection and seroconversion for each of the vaccine strains among the 3 groups of subjects. Results: Heroin users, subjects on MMT and non-user controls mount a similarly robust serologic response to TIV. GMT and rates of seroprotection and seroconversion were not significantly different among groups. Conclusion: Our results suggest that opioid use do not significantly alter antibody responses to influenza vaccine supporting the vaccination effort in these populations.
    Full-text · Article · Feb 2016
    • "Given the fact that miRNAs are involved in important biological processes and innate immunity against viral infections , including HCV ( Ding et al. 2012; Kawano et al. 2013), it is of significance to determine whether environmental factors such as heroin abuse, a common practice among IDUs, can dysregulate both cellular and cell-free miRNAs. Although it is known that opiates impair host immunity and enhance HCV replication in vitro (Li et al. 2003; Wang et al. 2005), there is little information about the in vivo impact of heroin on the expression of miRNAs related to innate immunity and HCV infection. "
    [Show abstract] [Hide abstract] ABSTRACT: Hepatitis C virus (HCV) infection is common among injection drug users (IDUs). There is accumulating evidence that circulating microRNAs (miRNAs) are associated with HCV infection and disease progression. The present study was undertaken to determine the in vivo impact of heroin use on HCV infection and HCV-related circulating miRNA expression. Using the blood specimens from four groups of the study subjects (HCV-infected individuals, heroin users with/without HCV infection, and healthy volunteers), we found that HCV-infected heroin users had significantly higher viral load than HCV-infected non-heroin users (p = 0.0004). Measurement of HCV-related circulating miRNAs in plasma showed that miRs-122, 141, 29a, 29b, and 29c were significantly increased in the heroin users with HCV infection, whereas miR-351, an HCV inhibitory miRNA, was significantly decreased in heroin users as compared to control subjects. Further investigation identified a negative correlation between the plasma levels of miR-29 family members and severity of HCV infection based on aspartate aminotransferase to platelet ratio index (APRI). In addition, heroin use and/or HCV infection also dysregulated a panel of plasma miRNAs. Taken together, these data for the first time revealed in vivo evidence that heroin use and/or HCV infection alter circulating miRNAs, which provides a novel mechanism for the impaired innate anti-HCV immunity among IDUs.
    Full-text · Article · Jan 2015
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