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Introduction
Hepatitis B virus (HBV) infection is a major cause of
chronic liver inflammation worldwide. Many chronic HBV
carriers suffer from progressive liver diseases, such as liver
cirrhosis (LC) and hepatocellular carcinoma (HCC) during
their lifetime. The outcome of hepatitis B patients is closely
linked to their immune status. Immunity plays a decisive role
in host-virus interactions, and greatly influences viral
replication and the clinical outcome of
infection[1]. Chronic HBV infection is associated with T cell
hyporesponsiveness or
tolerance[2]. Thus, innate and/or adaptive immune responses
are likely to be either absent or diminished when viral
persistence follows HBV infections.
Toll-like receptors (TLR) 7 and 9 are members of the
Toll-like family of receptors[3,4], and sense infection by detecting
molecular structures of invading microbial pathogens and
initiate innate immune responses[5]. These receptors
mediate adaptive immune responses by activating immune cells,
such as dendritic cells (DC) [4,6,7]. TLR7 and TLR9 are present
on both immune and non-immune cells[8]. TLR9 recognizes
unmethylated deoxycytidyl-phosphate-deoxyguanosine
dinucleotides, which are common in bacterial and some viral
nucleic acids[9], whereas TLR7 recognizes single-stranded
RNA in the cytoplasm of infected cells during viral
replication[10]. High expression levels of TLR9 were recently
detected in clinical samples of lung and breast cancer and
corresponding cell lines[11,12], suggesting a relationship between
TLR9 and carcinogenesis.
Recent studies on TLR9 subfamily members describe the
relationship between viral pathogens such as hepatitis C
virus (HCV), herpes simplex virus, HIV, and these
TLR[13_16]. It was demonstrated that the secretion of type I interferon
(IFN) in response to these viruses is mediated by the
TLR7/TLR9 pathway. However, the expression profiles of TLR7
and TLR9 in the peripheral blood mononuclear cells (PBMC)
of patients with chronic HBV infection and related HCC have
not been evaluated. Information about the regulation of
TLR by viruses is also
limited[10,17,18]. The current study was
undertaken to investigate the expressions of TLR7 and TLR9
in the PBMC of chronic HBV and related HCC patients, and
to analyze any relationship between the TLR expression and
the disease states.
Materials and methods
Study patients The study patients included 11 healthy
volunteers (normal controls, NC) and 41 patients at various
stages of chronic HBV infection, including 19 with chronic
hepatitis B (CHB), 11 with HBV-related LC, and 11 with
HBV-related HCC. The patients were enrolled at the First
Affiliated Hospital, School of Medicine, Zhejiang University
(Hangzhou, China) from February 2006 to May 2006.
Informed consent was obtained from each patient, and the study
protocol was approved by the Ethics Committees of the
School of Medicine, Zhejiang University. The clinical
parameters of the patients are shown in Table 1.
The diagnostic criteria conformed to "The guideline of
prevention and treatment for chronic hepatitis
B"[19]. The patients had not received any surgical treatment and had
not received any antiviral treatment in the last month. All LC
patients were in Child class B and C by Child-Pugh score
evaluation. HCC patients were confirmed by liver tissue
biopsy. All healthy volunteers were negative for HCV, HBV,
and HIV.
Preparation of PBMC The PBMC were separated from
5 mL heparinized whole blood by centrifugation on a
Lym-pholyte-H system (Cedarlane, Hornby, Canada) by gradient
centrifugation. The PBMC were resuspended in ACK lysis
buffer (0.15 mol/L NH4Cl, 1 mmol/L
KHCO3, 0.1 mmol/L Na2 EDTA, pH 7.4) to lyse the red blood cells.
RNA isolation, reverse transcription, and quantitative
real-time PCR Total RNA was extracted using Trizol
reagent (Invitrogen, Carlsbad, CA, USA) from
1×106~2×106 PBMC. Reverse transcription was performed using the
RevertAid first-strand cDNA synthesis kit (MBI
Fermentas,Vilnius, Lithuania). Real-time PCR was performed with the
FastStart DNA SYBR premix ex Taq kit (Gene Home
Biotechnology, Hangzhou, China) using an ABI 7500
system (Applied Biosystems, Foster, CA, USA). The primers
were synthesized by Shanghai Yingjun Biotech (Shanghai,
China) as follows: TLR7 forward, 5'-TGGAAATTGCCC TCGTTGTT-3', reverse,
5'-GTCAGCGCATCAAAAGCATT-3'; TLR9 forward, 5'-CCCGCTACTGGTGCTATCC-3', reverse,
5'-CCTTCCTCTTTCCACTCCC-3'; and β-actin forward,
5'-CCG-CCATGTAGGTCGCTAT-3', reverse,
5'-TGACACGCCATCA-CCAGAGT-3'. Thermocycling was performed at 95 °C for 20
s, followed by 40 cycles at 95 °C for 15 s, 58 °C for 10 s, and 72
°C for 40 s to measure the fluorescence signal. The
dissociation stages, melting curves, and quantitative analyses of the
data were performed using 7500 system SDS software v1.2.3
(Applied Biosystems, USA). The relative quantitation of
target gene expression was evaluated using the comparative
CT method as described by Ross et
al[20].
Western blot analysis The cytoplasmic extracts were
prepared by lysis in an lysis buffer containing 150 mmol/L
NaCl, 10 mmol/L Tris-HCl (pH 7.9), 0.5%Triton X-100, 0.6%
NP-40, and protease inhibitors (1 µg/mL leupeptin, 1 µg/mL
pepstatin A, and 2 µg/mL aprotinin). The protein contents
were determined using the DC protein assay kit (Bio-Rad,
Richmond, CA, USA). The PBMC lysates were mixed with
2×SDS sample buffer. In total, 40 μg of protein was
separated in a 10% polyacrylamide gel and transferred to a
polyvinylidene fluoride membrane (Millipore, Billerica, MA,
USA). After blocking with 5% skim milk powder in TBST,
the membrane was incubated with primary antibody, mouse
anti-TLR9 mAb or rabbit anti-TLR7 polyclonal antibodies
(Imgenex, San Diego, CA, USA) in TBST overnight at 4 °C.
Subsequently, the membranes were washed in washing buffer
(phosphate-buffered saline with 0.1% Tween-20) incubated
with horseradish peroxidase-conjugated secondary antibody,
rabbit antimouse, or goat anti-rabbit (Pierce, 1:10000 in
blocking buffer) for 1 h at room temperature. The reactive bands
were visualized with an EZ-ECL kit (Bioind, Kibbutz, Israel).
A monoclonal anti-β-actin antibody was used as a control at
a dilution of 1:400 (Santa Cruz, CA, USA).
Intracellular TLR staining and flow cytometry
analysis A total of
6×106_10×106 PBMC were fixed and permeabilized
with IntraPrep permeabilization reagent (Beckman Coulter,
Fullerton, CA, USA). The cells were then stained with
fluorescein-isothiocyanate-conjugated anti-TLR9 mAb or an
unconjugated rabbit anti-TLR7 polyclonal antibody (Imgenex, San Diego, CA, USA). PE-conjugated goat
antirabbit immunoglobulin G (Molecular Probes, Eugene, OR,
USA) was used as a secondary antibody for TLR7 staining.
Stained PBMC were analyzed on a FACSCalibur machine
(BD Biosciences, San Jose, CA, USA). Data were analyzed
using CellQuest software (BD Biosciences, San Jose, CA,
USA)
Serum HBV_DNA assay The viral load of HBV_DNA in
the serum samples was quantified using a high sensitivity
fluorescent real-time PCR kit (PG biotech, Shenzhen, China)
and amplified using a Light Cycler 2.0 instrument (Roche
Applied Science, Basel, Switzerland). The detection
sensitivity of the PCR assay was
4×102 copies/mL.
Statistical analysis Statistical analyses (non-parametric
tests, Mann-Whitney U-test) were performed using SPSS
software version 13.0 (SPSS, Chicago, IL, USA). Correlation
analyses were calculated according to the Spearman-Rho
method. A P-value of less than 0.05 was considered to be
significant.
Results
Decreased expression of TLR7 and TLR9 mRNA in PBMC of patients
As indicated in Figure 1, the patient group with the lowest TLR7 and TLR9 expressions in PBMC was
the CHB group. The expressions of TLR7 and TLR9 mRNA
decreased more than 23.2- and 7.6-fold, respectively,
compared with the NC group. Between the NC and HCC groups,
the decrease of the TLR7 mRNA expression was not
significantly different compared with the NC group
(P>0.05). The decrease in the TLR7 mRNA expression in the LC and HCC
groups differed 2.8- to -0.34-fold, respectively, compared to
the NC group (P>0.05). A similar but weaker trend was also
observed in the decrease of TLR9 mRNA expression among
the groups. The expression of TLR9 mRNA decreased in the
LC and HCC patients by more than 6- and 1.7-fold,
res-pectively, compared with the NC group.
Western blot analysis of TLR7 and TLR9 in PBMC
There was a significant decrease in the TLR7 expression of
the CHB group compared with controls, and an increased
TLR7 expression in various patient stages (Figure 2). In
contrast, the TLR9 expression significantly increased in the
3 patient groups compared with the NC group. An upward
tendency was observed in the CHB, LC, and HCC groups.
Flow cytometry analysis of TLR7 and TLR9 expressions
in PBMC Mean fluorescence intensity (MFI) values
corresponding to TLR7 were downregulated in all patient groups
compared with the control group (P<0.05, Figure 3). The
MFI±SEM values were: NC=196.6±14.2, CHB=70.1±3.9,
LC=79.7±3.7, and HCC=83.8±3.2. The results showed no
significant differences among the 3 patient groups
(P>0.05).
The MFI values corresponding to TLR9 increased in all
the patient groups compared with the NC group
(P<0.05). The MFI±SEM values were: NC=76.2±6.2, CHB=183.7±15.1,
LC=240.8±34.4, and HCC=250.2±45.5. The TLR9 expression
in the HCC patient group was upregulated (P<0.05)
compared to the CHB patient group.
Correlation between the TLR protein expression and
serum HBV_DNA levels The expression of the TLR7
protein was negatively correlated with the serum copies of
HBV_DNA (r=_0.669, P<0.01), and the upregulation of the TLR9
protein was positively correlated with the serum copies of
HBV_DNA (r=0.563, P<0.01).
Discussion
After recognizing particular microbial molecules, subsets
of immune cells in PBMC equipped with the
corresponding set of TLR release immunological repertoire, such as
IFN-α, TNF-α, and interleukin-6, which directly regulate
immunocompetent cells[8]. TLR7 and TLR9 are members of the TLR9
subfamily[21,22]. Recent research indicates that the ligands
for TLR3, TLR4, TLR5, TLR7, and TLR9 can inhibit HBV
replication in the livers of HBV transgenic
mice[23], suggesting that TLR7 and TLR9 may play a role in regulating HBV.
The high expression of TLR9 was recently detected in
clinical samples of lung and breast cancer and corresponding
cell lines[11,12], suggesting a possible role in carcinogenesis.
If TLR7 and TLR9 are associated with the recognition of
specific viral components, it is important to characterize TLR7
and TLR9 expression in the PBMC of patients with chronic
HBV infection and HBV-related HCC to further understand
pathogen-host interactions and predicted outcomes.
The results of our study clearly show decreased TLR7 at
the mRNA and protein levels, and TLR9 expression at the
mRNA level in patient PBMC compared with the healthy
controls. These results are different from the upregulated
TLR after other virus infections, as shown in previous
studies[24,25]. HBV may elicit a factor that inhibits TLR7
expression and TLR9 mRNA in the PBMC of patients, resulting in
immune escape and even immunological tolerance. Because
the same decreased TLR7 and TLR9 expressions of
plasmacytoid DC were also found in further studies, the alteration
of TLR7 and TLR9 expressions even impair the IFN-α
production of pDC (unpublished data).
The regulation of eukaryotic gene expression is the
result of complex, multi-level processes; mRNA levels do not
always correspond to the actual expressed protein
levels[26_28]. The increase of the TLR9 protein might be related to
post-transcriptional processes and might be involved in the
immune surveillance of HCC. This also may reflect negative
feedback inhibition from the TLR9 protein to the mRNA or
other regulatory influences which are not currently
understood[29].
TLR9 is mostly localized in the endoplasmic
reticulum[30] and traffics to endosomal and lysosomal compartments after
cellular activation[31,32]. Some studies also showed that a
fraction of the TLR9 expresses on the cell surface, in the
gastric epithelium[33], intestinal epithelial
cells[34], and some peripheral blood mononuclear
cells[35]. It was no doubt that FACS data indicated the functional cellular expression of
TLR while the Western blot analysis showed the total TLR
expression, including the superficial pool. These results
reflect another level of regulation that is protein trafficking.
The TLR9 expression in the HCC group was the highest,
which may be directly related to carcinogenesis of HCC. The
statistical analysis indicated no difference in the TLR9
levels among the HCC and LC groups. If the sample size was
enlarged, the results may be different. The expression of
TLR7 was not different among the groups of patients,
suggesting that unlike TLR9, TLR7 has no correlation with HCC.
Finally, we were interested in understanding whether
changes in TLR7 and TLR9 expressions were correlated with
the levels of serum HBV_DNA. Positive results suggested
that the TLR expression was related to the state of HBV
replication. We conclude that HBV might produce an
inhibitor when it is replicating. However, the expression of TLR9
was influenced by chronic HBV infection and the
carcinogenesis of HCC, which may further complicate the situation.
In summary, this study is the first investigation of the
expression of TLR7 and TLR9 of PBMC in CHB, LC, and
HCC patient groups. A larger scale clinical investigation
should be undertaken to determine whether TLR7 and TLR9
expressions may be used to evaluate patient immunity and
predict HCC outcomes.
Acknowledgements
We thank Prof Yong-min TANG for his expert technical
assistance with the flow cytometry. We are grateful to Prof
Fang XU and Dr Ping LI for reviewing this manuscript and
for their valuable advice.
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