Dept of Infectious DiseasesShanghai Ruijin Hospital

Jiaotong University School of MedicineQing Xie

Pattern Recognition Receptors and HBV Infection

Role of Toll-like Receptors

HBsAg HBsAg raterate (%) (%)

8:8: highhigh

2-7:2-7: mediummedium

<2:<2: lowlow

HBV infection is still a major global health care problem

• 350 million chronic carriers worldwide• 9th leading causes of death• 75% of HBV carrier are Asian

High prevelance

Widely spread

Nature History of HBV InfectionNature History of HBV Infection

AcuteHBVInfection

Chronic HBVInfection

ChronicHepatitis B

Cirrhosis

Liver Failure

HCC

3-5% Adult3-5% Adult

95% Children95% Children

5 年

6-15%5 年

20-23%5 年

12-20%

Antiviral treatment

HBV Virons

Complete Response Partially Response 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

HOST

Virus clearance

resolved

Unable to clear virus

Persistent infection

Progression of disease

Immune function of Host

Central Role

Three essential elements required in liver Three essential elements required in liver inflammation following HBV infectioninflammation following HBV infection

HBV VIRONS

HEPATOCYTES

IMMUNOCYTES

Non antigenNon antigen specific-specific-INNATEINNATE

IMMUNITYIMMUNITY

NK/TNK/T

PhagocytesPhagocytes

NKNK

pDCpDC

Plasmacytoid DC

调节性 T细胞

Antigen specific-Antigen specific-ADAPTIVEADAPTIVEIMMUNITYIMMUNITY

CD8CD8

CD4CD4

BB

Tregs CD4+CD25+CD4+CD25+Treg cellTreg cell

LINKLINK

CytokinesCytokinesType I interferon Type I interferon

IL-12IL-12Granzyme BGranzyme B

mDCMyeloid DC

AntigenAntigenpresentationpresentation

Th17Th17

Host

Virus Clearance

Host Immune Response is mediated by PattPatt

ern Recognition Receptors(PRR)ern Recognition Receptors(PRR) which reco

gnizes specific molecular or replication inte

rmediator of virus particles.

HBV VironsType I IFNInduction

Activated Cytotoxic Response

Pattern Recognition Receptors(PRR)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??

HBVG+bacteria G-bacteria

Virus clearance

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.

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.

Requirement of DC involment in TLR-initiated antivirus response

Distribution of TLR on DCsDistribution of TLR on DCs mDC ： （ myeloid dendritic cell ， mDC ） （ CD3 、 CD14 、 CD16 、 CD19 、 CD20 、 CD56 ） － BDCA1(CD1c)+ CD11c+

TLR1 TLR2 TLR3 TLR4 TLR5 TLR6 TLR8pDC ：（ 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?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??

CHB ： 28 cases Healthy ： 22 cases

Study Populations

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

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

0

25

50

75

100

125

patients n=28

controls n=18

p>0.05p

osi

tive

ce

ll %

0

100

200

300

400

500

600

p<0.05

patients n=28

controls n=18

MF

I

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.

A B C

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).﹦

A B

0.0 0.1 0.2 0.3 0.4 0.50

100

200

300

400

500

600

r =-0.646 p=0.02

pDC/PBMC%

ALT

??(I

U/L

)

CHB NS0.00

0.25

0.50

0.75

n=21 n=26

p<0.05

pD

C f

req

ue

ncy

%

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.

A B C

0

500

10001500

2000

2500

30003500

4000

4500

n=22 n=22PBMC PBMC-pDC

IFN

a(p

g/L

)

0

1000

2000

3000p>0.05

patients n=22

controls n=20

IL-6

pg

/ml

0

10000

20000

30000

patients n=22

controls n=20

p<0.05

IFN

-a p

g/m

l

A B C

0

1000

2000

patients n=22

controls n=20

p>0.05

TN

F p

g/m

l

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 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 pa

tients with CHB.

2. pDCs are functionally impaired with the lower ability to pr

oduce IFN-α in patients, that may partly contribute to hep

atitis B evading an adequate immune response, resulting in

HBV persistent infection in host.

3. HBsAg and HBcAg were detectable in pDCs of patients s

uggest that functional impairment of pDCs may correlate w

ith HBV infection of pDCs.

60

80

100

0h 12h 24h 48h

Ti me

The

expr

essi

ons

ofTL

R3(%

)

CHHV

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 。

0h

48h24h

12h 0h

48h24h

12h

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 。

00. 050. 1

0. 150. 2

0. 250. 3

0. 350. 4

0. 450. 5

0h 12h 24h 48h

CHHV

*

*

0

0. 2

0. 4

0. 6

0. 8

1

1. 2

0h 12h 24h 48h

CHHV

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) 。

*

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) 。

*

00. 020. 040. 060. 080. 1

0. 120. 140. 160. 18

0h 12h 24h 48h

CHHV

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.

*

020

4060

80100

120140

160180

200

0h 12h 24h 48hTi me

The IFN-beta(pg/ml)

CHHV

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 ds

RNA 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

CONCLUSIONS