1. 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

2. HBV infection is still a major global
health care problem
High prevelance
Widely spread
HBsAg rate (%)
8: high
2-7: medium
<2: low
350 million chronic carriers worldwide
9th leading causes of death
75% of HBV carrier are Asian

3. Nature History of HBV Infection
HCC
HBV Virons 5年
6-15%
3-5% Adult
12-20%
Acute
HBV
Infection
Chronic
HBV
Infection
Chronic
Hepatitis B
Cirrhosis 5年
95% Children 5年
20-23%
Antiviral treatment
Liver Failure
Complete Response
Partially Response
Non Response
Immune as a key factor influences the outcome and progression of disease

4. Challenge: What mechanism of virus clearance of host??
How to do that??
Virus
clearance
resolved H O S T
Central
Role
HBV
Unable to
clear virus
Immune function
of Host
Persistent infection
Progression of disease

5. Three essential elements required in liver
inflammation following HBV infection
HBV VIRONS
IMMUNOCYTES
HEPATOCYTES

6. Non antigen specific-INNATE
IMMUNITY
NK/T
Phagocytes NK
pDC
Plasmacytoid DC
Antigen specific-ADAPTIVE
IMMUNITY
LINK
Cytokines
Type I interferon
IL-12
Granzyme B
mDC
Myeloid DC
Antigen
presentation
CD8 B
Th17
CD4
Tregs
CD4+CD25+
Treg cell
调节性T细胞

7. Host HBV Virons Type I IFN Induction Activated Cytotoxic Response
Virus
Clearance
Activated Cytotoxic
Response
Host Immune Response is mediated by Pattern Recognition Receptors(PRR) which recognizes specific molecular or replication intermediator of virus particles.

8. Pattern Recognition Receptors(PRR)

9. 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.

10. G+bacteria
G-bacteria
Virus
clearance

11. What is a role of TLR in HBV infection??
G+bacteria
G-bacteria
Disturbance of
TLR expression
induces the persistent
infection.
Abnormal of signal passway
initiated by TLR induces the
inability to clear HBV or
overclearance, that leads to
immunotolerance or immune
injury.
Virus
clearance

12. 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.

13. 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

14. 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??

15. 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.

16. Selection and preparation of pDC、mDC
BDCA4 DC isolation kir
mDC
BDCA1 DC siolation kit

17. p>0.05
p<0.05
125
600
100
500 75
400
positive cell %
MFI
300 50
200 25
100
patients
controls
patients
controls
n=28
n=18
n=28
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

18. 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 marker-negative fraction (R2), (C) the CD123/BDCA-2-double positive represents the plasmacytoid dendritic cells.

19. A B
p<0.05
0.0
0.1
0.2
0.3
0.4
0.5
100
200
300
400
500
600
r = p=0.02
pDC/PBMC%
ALT??(IU/L)
0.75
0.50
pDC frequency%
0.25
0.00
CHB NS
n=21
n=26
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).

20. A B C
4500
4000
3500
3000
2500
IFNa(pg/L)
2000
1500
1000
500
PBMC
PBMC-pDC
n=22
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.

21. A B C
10000
20000
30000
patients
n=22
controls
n=20
p<0.05
IFN-a pg/ml
1000
2000
patients
n=22
controls
n=20
p>0.05
TNF pg/ml
1000
2000
3000
p>0.05
patients
n=22
controls
n=20
IL-6 pg/ml
Fig.7. Cytokine production by isolated peripheral precursor pDCs of chronic HBV patients (n=22) and healthy controls (n=20) after stimulation with CpG-ODN 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.

22. The decrease of TLR9 expression ability by DC is related to HBV infection in DC？
Hepatology 2006;43(3):
HBsAg
HBcAg

23. 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.

24. 0h
12h 0h
12h
24h
48h
24h
48h
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。

25. * *
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。

26. *
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)。

27. *
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)。

28. *
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.

29. 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