单击此处编辑母版标题样式,*,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,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,HBsAg rate(%),8:high,2-7:medium,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 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 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=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-negative fraction(R2),(C)the CD123/BDCA-2-double positive represents the plasmacytoid dendritic cells.,A,B,C,Fig.5.AIn 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,0.0,0.1,0.2,0.3,0.4,0.5,0,100,200,300,400,500,600,r=-0.646 p=0.02,pDC/PBMC%,ALT?(IU/L),CHB,NS,0.00,0.25,0.50,0.75,n=21,n=26,p0.05,patients,n=22,controls,n=20,IL-6 pg/ml,0,10000,20000,30000,patients,n=22,controls,n=20,p0.05,TNF 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 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 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 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 impairment 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 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 was observed at 48 hrs.*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 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.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 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,