Transcript (P>0.05)。

Pattern Recognition Receptors
and HBV Infection
Role of Toll-like Receptors
Dept of Infectious Diseases
Shanghai Ruijin Hospital
Jiaotong University School of Medicine
Qing Xie
HBV infection is still a major global
health care problem
High prevelance
Widely spread
• 350 million chronic carriers worldwide
• 9th leading causes of death
• 75% of HBV carrier are Asian
HBsAg rate (%)
8: high
2-7: medium
<2: low
Nature History of HBV Infection
HCC
HBV Virons
5年
3-5% Adult
Chronic
HBV
Infection
Acute
HBV
Infection
Chronic 12-20%
Cirrhosis
Hepatitis
B
5年
95% Children
5年
Antiviral treatment
Complete Response
Partially Response
6-15%
20-23
Liver
Failure
Non Response
Immune as a key factor influences the outcome and
progression of disease
Challenge:
What mechanism of virus clearance of host??
How to do that??
HBV
Immune function
of Host
Virus
clearance
H
O
S
T
resolved
Unable to
clear virus
Central
Role
Persistent infection
Progression of disease
Three essential elements required in liver
inflammation following HBV infection
HBV VIRONS
IMMUNOCYTES
HEPATOCYTES
Non antigen specificINNATE
IMMUNITY
Plasmacytoid DC
pDC
Antigen specificADAPTIVE
IMMUNITY
LINK
Cytokines
Type I interferon
IL-12
Granzyme B
CD8
Antigen
presentation
Phagocytes
mDC
NK/T
B
Myeloid DC
Th17
NK
Tregs
调节性T细胞
CD4+CD25+
Treg cell
CD4
HBV Virons
Host
Type I IFN
Induction
Activated Cytotoxic
Response
Virus
Clearance
Host Immune Response is mediated by
Pattern Recognition Receptors(PRR) which
recognizes specific molecular or replication
intermediator of virus particles.
Pattern Recognition Receptors(PRR)
Recent Studies found:
 The close relationship between host
innate immunity and the recognition
of pathogen and clearance.
 TLR as an important PRR,plays a
central role in defence of virus
invasion.
 Different TLR can recognize various
pathogen.
G+bacteria
G-bacteria
Virus
Virus
Virus
clearance
What is a role of TLR in HBV
infection??
HBV
G+bacteria
G-bacteria
 Disturbance of
TLR expression
induces the persistent
infection.
Virus
clearance
 Abnormal of signal passway
initiated by TLR induces the
inability to clear HBV or
overclearance, that leads to
immunotolerance or immune
injury.
Requirement of DC involment in
TLR-initiated antivirus response
 Toll-like receptor (TLR)-mediated recognition of specific
structures of invading pathogens initiates innate as
well as adaptive immune responses via antigen-presenting
cells (APCs).
 DC represents the most potent antigen-presenting cells
and thus plays an important role in the induction of innate
and specific immune response.
Distribution of TLR on DCs
mDC： （myeloid dendritic cell，mDC）
（ CD3、CD14、CD16、CD19、CD20、CD56）
－BDCA1(CD1c)+ CD11c+
TLR1 TLR2 TLR3 TLR4 TLR5 TLR6 TLR8
pDC：（plasmacytoid dendritic cell,
pDC）
（ CD3、CD14、CD16、CD19、CD20、
CD56）－BDCA2+ BDCA4+ CD123+
TLR7 TLR9
TLR3、TLR7、TLR9 参与抗病毒免疫
Scientific Questions?
 Function
of Myeloid and
Plasmacytoid Dendritic Cells of
Patients with CHB??
 Expression
of TLR-3, TLR-9 in
patients following HBV chronic
infection??
Study Populations
CHB： 28 cases
Healthy：22 cases
Including criteria：
（1） Serum HBsAg positive > 6 months.
（2） Abnormal liver function for 2 times with 2 weeks apart，
ALT>1.5 xU/L at screeing.
（3） Serum HBeAg(+)，HBeAb(-).
（4） Serum HBV-DNA>1×105 /L.
（5） excluded liver cirrhosis by liver ultrasound
（6） excluded HIV、HCV、HDV、HEV coinfection.
（7） Not allowed to use antiviral treatment or immunmodulator
within 6 months.
Selection and preparation of
pDC、mDC
pDC
BDCA4 DC
isolation kir
mDC
BDCA1 DC
siolation kit
p<0.05
p>0.05
positive cell %
125
600
500
400
300
200
100
0
100
MFI
75
50
25
0
patients
n=28
controls
n=18
patients
n=28
controls
n=18
Fig.3. TLR9 expression of isolated peripheral precursor pDCs of
chronic HBV patients (n=28) and healthy controls (n=18). (A) No
difference in the percentage of pDCs expressing TLR9 was found
between the patients and the controls. (B) The MFI of TLR9 from
patients were significantly reduced compared to controls. Data are
expressed as mean±SD
A
B
C
Fig.4. Flow cytometric analysis for enumeration of pDCs. PBMCs were isolated
and stained with the following antibodies：Lin-FITC( CD3,CD14,
CD16,CD19,CD20,CD56), PE-BDCA-2, and APC-CD123. (A) Dead cells were
excluded by forward side scatter analysis. (B) After gating on the lineage markernegative fraction (R2), (C) the CD123/BDCA-2-double positive represents the
plasmacytoid dendritic cells.
A
B
p<0.05
600
r =-0.646 p=0.02
500
0.50
ALT??(IU/L)
pDC frequency%
0.75
0.25
400
300
200
100
0
0.00
CHB
n=21
NS
n=26
0.0
0.1
0.2
0.3
0.4
0.5
pDC/PBMC%
Fig.5. （A）In patients with CHB the relative numbers of pDCs were
significantly reduced compared healthy controls(P<.05). (B) Correlation
of the relative frequency of pDCs with the ALT levels in chronic HBV
infection. The relative counts of pDCs were inversely correlated with ALT
values ( P<.05; r﹦-0.645).
B
C
IFNa(pg/L)
A
4500
4000
3500
3000
2500
2000
1500
1000
500
0
PBMC
n=22
PBMC-pDC
n=22
Fig.6. pDCs were the main source of IFN-αin the peripheral blood of
humans. (A) An example of frequency analysis of pDCs in PBMCs. (B)
PBMCs were analyzed by flow cytometer which showed pDCs were almost
depleted. (C) PBMCs were separated with magnetic beads coupled to
BDCA-4 antibodies and both fractions were stimulated with CpG-ODN 2216 ,
which show great amount of IFN-αin the supernatant of the BDCA-4-positive
fraction compared to the BDCA-4-negative fraction.
C
B
A
p<0.05
p>0.05
p>0.05
3000
2000
10000
0
2000
IL-6 pg/ml
20000
TNF pg/ml
IFN-a pg/ml
30000
1000
1000
0
0
patients
n=22
controls
n=20
patients
n=22
controls
n=20
patients
n=22
controls
n=20
Fig.7. Cytokine production by isolated peripheral precursor pDCs of chronic
HBV patients (n=22) and healthy controls (n=20) after stimulation with CpGODN 2216. After 24 hours of stimulation, cytokine production was determined in the
culture supernatants by specific ELISAs. (A) pDCs of patients were significantly
impaired in their ability to produce IFN-αcompared to healthy controls. No difference
was detected in the production of (B) TNF-αand (C) IL-6 between patients and
healthy controls.
The decrease of TLR9 expression ability by
DC is related to HBV infection in DC？
Hepatology 2006;43(3):539-547
HBsAg
HBcAg
CONCLUSIONS
1. The TLR9 expression of circulating pDCs is reduced in
patients with CHB.
2. pDCs are functionally impaired with the lower ability to
produce IFN-α in patients, that may
partly contribute to
hepatitis B evading an adequate immune response,
resulting in HBV persistent infection in host.
3. HBsAg and HBcAg were detectable in pDCs of patients
suggest that functional impairment of pDCs may correlate
with HBV infection of pDCs.
12h
0h
24h
48h
24h
The expressions of
TLR3(%)
0h
12h
48h
100
CH
HV
80
60
0h
12h
24h
48h
Time
Fig8. The levels of TLR3 expression on mDC of peripherial blood
between contrl and patients. It shows that TLR3 level in control is
increased following 24 hrs stimulaton, however the increase of TLR3
expression was delayed to 48 hrs following stimulation. P<0.05。
0.5
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
CH
HV
*
*
0h
12h
24h
48h
Fig9. The changes of TLR3 mRNA by real-time PCR at various
timepoints following stimulation.It shows TLR3 mRNA is
increased significantly at 12 hrs, it recovers to the
baseline at 24 hrs and 48 hrs. However the increase of TLR3
mRNA in CHB patients was observed at 48 hrs. *P<0.05。
1.2
1
0.8
CH
HV
0.6
*
0.4
0.2
0
0h
12h
24h
48h
Fig10. Expression of TBK1 mRNA on mDC by RT-PCR. There is no
difference in TBK1 mRNA at baseline between control and
patients (P>0.05)。TBK1 mRNA is increased significantly at 12
hrs when compared to baseline in control (P<0.05). There is no
difference in TBK1 mRNA at 12hrs, 24hrs and 48hrs when
conpared to baseline (P>0.05)。
0.18
0.16
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
CH
HV
*
0h
12h
24h
48h
Fig11. Expression of IFN-βmRNA by RT-PCR in mDC. There is no
difference in at baseline between control and patients (P>0.05)。
The level of IFN-βmRNA expression at 12 hrs is higher than at
baseline in control (P<0.05). However there is no deifference
between at various time points(P>0.05)。
The IFN-beta(pg/ml)
200
180
160
140
120
*
100
80
CH
HV
60
40
20
0
0h
24h
12h
48h
Time
Fig12. The detection of IFN-β by ELISA in supernatant on mDC following
polyI:C stimulation.The level of IFN-β at baseline is very similar
between two groups.There is no difference at various timepoints
following stimulation in patients.However,the level of IFN-βis much
higher at 12 hrs when compared to baseline.Also the difference was
observed at 12hrs, 24hrs and 48 hrs between control and patients.
CONCLUSIONS

The increase of expression level of TLR3 is slower and
delayed in CH groups than HV groups. It contributes to
the inability for the host to clear HBV.

The level of TBK1 molecular is quite low even following
dsRNA stimulation, suggesting that abnormal of signal
transduction passway may exist in HBV .

Our results suggest that dysfunction of TLR3 might play
an important role in chronic HBV infection. It may
provide new insights for understanding the mechanism
of persistent HBV infection