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1、Dept of Infectious DiseasesShanghai Ruijin Hospital Jiaotong University School of MedicineQing 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 worldwide9th leading causes of death75
2、%of HBV carrier are Asian,High prevelance,Widely spread,Nature History of HBV Infection,AcuteHBVInfection,Chronic HBVInfection,ChronicHepatitis B,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 Response,Non Res
3、ponse,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
4、elements required in liver inflammation following HBV infection,HBV VIRONS,HEPATOCYTES,IMMUNOCYTES,调节性T细胞,Antigen specific-ADAPTIVEIMMUNITY,CD8,CD4,B,Th17,Host,Virus Clearance,Host Immune Response is mediated by Pattern Recognition Receptors(PRR)which recognizes specific molecular or replication int
5、ermediator of virus particles.,HBV Virons,Type I IFNInduction,Activated Cytotoxic Response,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 defenc
6、e of virus invasion.Different TLR can recognize various pathogen.,What is a role of TLR in HBV infection?,HBV,G+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 overcle
7、arance,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 pl
8、ays 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 DCs,mDC：（myeloid dendritic cell，mDC）（CD3、CD14、CD16、CD19、CD20、CD56）BDCA1(CD1c)+CD11c+TLR1 TLR2 TLR3 TLR4 TLR5 TLR6 TLR8pDC：（plasmacytoi
9、d 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 Healthy：22 cases,S
10、tudy 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、HDV、HEV coinfection.（7）Not allow
11、ed 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 perc
12、entage 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%,0,100,200,300,400,500,600,p0.05,patients,n=
13、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 scatter analysis.(B)After gating on the lineage marker-
14、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 c
15、hronic 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 humans.(A)An example of frequency analysis of pDCs in PBMCs.(B)PBM
16、Cs 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-in the supernatant of the BDCA-4-positive fraction compared to the BD
17、CA-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 healthy controls(n=20)after stimulation with CpG-ODN 2216.After 24 hours of
18、 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 was detected in the production of(B)TNF-and(C)IL-6 between patients and healthy co
19、ntrols.,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-in patients,
20、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.,Fig8.The levels of TLR3 expression on
21、 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.P0.05。,0h,48h,24h,12h,0h,48h,24h,12h,Fig9.The changes of TLR3 mRNA by real-time PCR a
22、t 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.*P0.05。,*,*,Fig10.Expression of TBK1 mRNA on mDC by RT-PCR.There is no differen
23、ce 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-PCR in mDC.There is no difference in at baseline between control and patients(P0.05)。The level of IFN-mRNA
24、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 is very similar between two groups.There is no difference at various timepoints following stimulation in patients.
25、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 expression level of TLR3 is slower and delayed in CH groups than HV groups.It contributes to the inability for the ho
26、st 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,
