1、Dept of Infectious Diseases Shanghai Ruijin Hospital Jiaotong University School of Medicine Qing Xie,Pattern Recognition Receptors and HBV Infection,Role of Toll-like Receptors,HBV infection is still a major global health care problem,350 million chronic carriers worldwide 9th leading causes of deat
2、h 75% of HBV carrier are Asian,High prevelance,Widely spread,Nature History of HBV Infection,Acute HBV Infection,Chronic HBV Infection,Chronic HepatitisB,Cirrhosis,Liver Failure,HCC,3-5% Adult,95% Children,5年,6-15%,5年,20-23%,5年,12-20%,Antiviral treatment,HBV Virons,Complete Response,Partially Respon
3、se,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,H O S T,Virus clearance,resolved,Unable toclear virus,Persistent infection,Progression of disease,Immune function of Host,CentralRole,Thr
4、ee essential elements required in liver inflammation following HBV infection,HBV VIRONS,HEPATOCYTES,IMMUNOCYTES,调节性T细胞,Antigen specific-ADAPTIVE IMMUNITY,CD8,CD4,B,Th17,Host,VirusClearance,Host Immune Response is mediated by Pattern Recognition Receptors(PRR) which recognizes specific molecular or r
5、eplication intermediator of virus particles.,HBV Virons,Type I IFN Induction,Activated Cytotoxic Response,Pattern Recognition Receptors(PRR),Recent Studies found:,The close relationship between hostinnate immunity and the recognition of pathogen and clearance.TLR as an important PRR,plays a central
6、role in defence of virus invasion.Different TLR can recognize various pathogen.,What is a role of TLR in HBV infection?,HBV,G+bacteria,G-bacteria,Virusclearance,Disturbance of TLR expression induces the persistentinfection.Abnormal of signal passwayinitiated by TLR induces theinability to clear HBV
7、or overclearance, that leads to immunotolerance or immuneinjury.,Toll-like receptor (TLR)-mediated recognition of specificstructures of invading pathogens initiates innate aswell as adaptive immune responses via antigen-presentingcells (APCs).DC represents the most potent antigen-presenting cellsand
8、 thus plays an important role in the induction of innateand specific immune response.,Requirement of DC involment in TLR-initiated antivirus 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：
9、（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?,CHB： 28 cases
10、 Healthy：22 cases,Study Populations,Including criteria： （1） Serum HBsAg positive 6 months. （2） Abnormal liver function for 2 times with 2 weeks apart，ALT1.5 xU/L at screeing. （3） Serum HBeAg(+)，HBeAb(-). （4） Serum HBV-DNA1105 /L. （5） excluded liver cirrhosis by liver ultrasound （6） excluded HIV、HCV、
11、HDV、HEV coinfection. （7） Not allowed to use antiviral treatment or immunmodulatorwithin 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 control
12、s (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 meanSD,0,25,50,75,100,125,patients,n=28,controls,n=18,p0.05,positive cell %,
13、0,100,200,300,400,500,600,p0.05,patients,n=28,controls,n=18,MFI,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 scat
14、ter 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
15、 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,CHB,NS,0.00,0.25,0.50,0.75,n=21,n=26,p0.05,pDC frequency%,Fig.6. pDCs were the main source of IFN-in the peripheral blood of hu
16、mans. (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 I
17、FN-in the supernatant of the BDCA-4-positive fraction compared to the BDCA-4-negative fraction.,A,B,C,0,500,1000,1500,2000,2500,3000,3500,4000,4500,n=22,n=22,PBMC,PBMC-pDC,IFNa(pg/L),IL-6 pg/ml,A,B,C,Fig.7. Cytokine production by isolated peripheral precursor pDCs of chronic HBV patients (n=22) and
18、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-compared to healthy controls. No difference
19、was detected in the production of (B) TNF-and (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
20、CHB. 2. pDCs are functionally impaired with the lower ability to produce IFN- 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 impa
21、irment of pDCs may correlate with HBV infection of pDCs.,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
22、stimulation. P0.05。,0h,48h,24h,12h,0h,48h,24h,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 w
23、as observed at 48 hrs. *P0.05。,*,*,Fig10. Expression of TBK1 mRNA on mDC by RT-PCR. There is no difference in TBK1 mRNA at baseline between control and patients (P0.05)。TBK1 mRNA is increased significantly at 12 hrs when compared to baseline in control (P0.05)。,*,Fig11. Expression of IFN-mRNA by RT-
24、PCR in mDC. There is no difference in at baseline between control and patients (P0.05)。The level of IFN-mRNA expression at 12 hrs is higher than at baseline in control (P0.05)。,*,Fig12. The detection of IFN- by ELISA in supernatant on mDC following polyI:C stimulation.The level of IFN- at baseline i
25、s very similar between two groups.There is no difference at various timepoints following stimulation in patients.However,the level of IFN-is much higher at 12 hrs when compared to baseline.Also the difference was observed at 12hrs, 24hrs and 48 hrs between control and patients.,*,The increase of exp
26、ression 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,CONCLUSIONS,
