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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