Impacts Of Intrahepatic TLR3 Signaling Activation At The Early Stage Of HBV Infection And Its Mechanism

Objective To explore the impacts of intrahepatic TLR3 signaling activation at the early stage of HBV infection and its mechanism.Then to provide a possible experimental and theoretical basis for elucidating the persistent replication of virus in a small number of adults infected with HBV.Methods 1.Poly(I:C)or NS was co-applied with HBV plasmid p AAV-HBV1.2 by hydrodynamic injection(HI)into the tail veins of 8w,wild-type male BALB/C mice.2.HBV plasmid p AAV-HBV1.2 was applied by HI into the tail veins of BALB/C mice.After 10 days of plasmid p AAV-HBV1.2 injection,mice were treated with Poly(I:C)or NS by HI.3.Serum samples and liver tissues were harvested at regular time points.4.The levels of HBs Ag and HBe Ag in serum were detected by ELISA,the titers of virus in serum and liver were detected by Real-Time PCR.Liver sections were stained for hepatitis B c antigen(HBc Ag).The expression levels of HBc Ag were defined by immunohistochemistry.5.The liver infiltrates lymphocytes(LILs)were separated,then we detected IFN-γ producing LILs via an enzyme-linked immunospot assay(ELISPOT).6.Total RNA was extracted from mouse liver tissue,and the m RNA levels of CD3、CD4、CD8、 IL-6、IL-10、TGF-β、IFN-β、IFN-γ、ISG15、IP-10、PDL-1、IRF3、OAS、 Perf、 Fox P3 were measured by Real-time RT-PCR.7.The isolation of non-parenchymal liver cells(NPC and hepatocytes were performed by using Liberase Blendzymes.The isolation of liver sinusoidal endothelial cells(LSECs),and Kupffer cells(KCs)were performed by using Anti-CD146 or Anti-F4/80 Micro Beads.The RNA of different cells were extracted,and the m RNA levels of CD3、CD4、CD8、 IL-6、 IL-10、 TGF-β、 IFN-β、 IFN-γ、 ISG15、 IP-10、PDL-1、 IRF3、 OAS、 Perf、 Fox P3 were measured by Real-time RT-PCR.8.The NPCs and spleen cells were isolated and the frequency of various immune cells in NPCs and splenocytes were detected by flow cytometry after staining with CD49b、CD19、CD11b、CD11c、F4/80、CD4、CD25 and Fox P3.9.IFNAR1 antibody,PAAV-HBV1.2 plasmid and poly(I:C)were used at the same time by HI into the tail veins of BALB/C mice to block type Ⅰ interferon signaling pathway.And we analyzed the relationship between type Ⅰ interferon and persistent HBV replication in this mouse model.10.Anti-CD25 antibody was used by intraperitoneal injection on day-3 and 3 after injection of PAAV-HBV1.2 plasmid and poly(I:C)in BALB/C mice to neutralizing Treg cells.Anti-IL-10 receptor antibodies were injected by intraperitoneal injection on day-1,6,13 and 20 after injection of PAAV-HBV1.2 plasmid and poly(I:C)in BALB/C mice to block the IL-10 signaling pathway.And we analyzed the relationship between Treg/IL-10 and persistent HBV replication in this mouse model.11.Figures were made by Graphpad6.0 software.Statistical analyses were performed using SPSS18.0 statistics software.Differences between groups were compared using t-test.Data are expressed as standard error of the mean(SEM),and data were considered statistically significant when values reached P<0.05.Significance was denoted as P<0.05,P<0.01.Results 1.Activation of intrahepatic TLR3 signaling by HI at the early stage of infection leads to HBV persistence.In 80% of control mice,serum HBs Ag,HBe Ag and HBV DNA were completely eliminated on the 40 th day after HBV plasmid injection,intrahepatic HBc Ag and HBV DNA were also negative on the 40 th day.In the Poly(I:C)-treated mice,serum HBs Ag and HBV DNA were still positive on the 60 th day after HBV plasmid injection,and intrahepatic HBc Ag and HBV DNA were also positive on the 60 th day.2.After activation of intrahepatic TLR3 signaling by HI at the early stage of infection,the frequencies of anti-specific IFN-γ secreting LILs were significantly reduced on the 15 th day.3.After activation of intrahepatic TLR3 signaling by HI at the early stage of infection,the expression m RNA levels of IFN-b,IP10,OAS and ISG15 in liver tissue were up-regulated on the 1th day after injection,and the expression of IL-10 was up-regulated from 1th to 20 th day.The expression of CD4 was down-regulated on 1th,40 th and 60 th day,and the expression of CD8 was down-regulated on 1th,10 th and 40 th day.After activation of intrahepatic TLR3 signaling by HI at the early stage of infection,the expression of IFN-b m RNA in hepatocytes and LSEC was up-regulated on 1th day,and the expression of IL-10 m RNA in KC and remaining cells was up-regulated on the 15 th day.4.After activation of intrahepatic TLR3 signaling by HI at the early stage of infection,Poly(I:C)induced significant increases in NK cells,DCs and decreases in B cells and KCs in liver 1 days after infection.In contrast,the changes of cell numbers in spleen are just the opposite.After 15 days of Poly(I:C)infection,NK cells,KCs and Tregs in liver were increased.We also detected increases in NK cells and decreases in macrophagocytes in spleen 15 days post HI.Significantly more CD4+Foxp3+ Treg cells were detected in LILs of mice receiving poly(I:C)in comparison to the NS control group 7 and 15 days dpi.5.After IFNR1 blockade,HBs Ag,HBe Ag,and HBV DNA in mouse serum could all be cleared on the 60 th day after injection,while HBs Ag and HBV DNA in serum of control group were both positive on the 60 th day.6.After IFNR1 blockade,the frequencies of anti-specific IFN-γ secreting LILs were increased on 20 th day.7.After IFNR1 blockade,the expression of OAS and ISG15 m RNA in liver tissue were down-regulated on 1th day and the expressions of IFN-b and IL-10 m RNA were down-regulated on 20 th day.The expression of CD3 and CD8 m RNA were up-regulated on 10 th day and the expression of CD4 m RNA was up-regulated on 20 th day.The expression of IFN-β was down-regulated in hepatocytes and LSECs 1 day after IFNR1 blockade.8.IFNR1 blockade induced a numerical increase in many immune subsets in liver 1 days after infection including the total number of NK cells,DCs,B cells,monocytes and KCs.15 days after IFNR1 blockade,the intrahepatic Treg cells were significantly increased.The number of B cells increased,and the DC cells,monocytes and Treg cells decreased in the spleen on 15 th days.And the numbers of CD4+Foxp3+ Treg cells were decreased 15 days after IFNR1 blockade.9.Poly(I:C)was injected 10 days after PAAV-HBV1.2 plasmid injection,HBs Ag and HBe Ag in the serum of mice can be completely eliminated on the 4th day after Poly(I:C)injection,while the control group is still positive.On the 4th day,the number of intrahepatic CD8+ T cells increased significantly,and the number of intrahepatic B cells increased significantly on the 15 th day.10.After blocking Treg or blocking the IL-10 signaling pathway,serum HBs Ag in mice can be cleared on the 40 th day after injection,while serum HBs Ag in the control group is positive on the 60 th day.Conclusion 1.Activation of intrahepatic TLR3 signaling at the early stage of HBV infection led to HBV persistence in mice model through the up-regulation of hepatic IL-10 expression,and an increase in the number of Treg cells and down-regulates virus-specific T cell responses.2.Intrahepatic TLR3/IFN pathway is activated at the late stage of HBV infection,CD8+ T cells are rapidly recruited to the liver together with B cells recruited,may participate in the clearance of HBV 3.Activation of intrahepatic TLR3 pathway at different time points has different effects on the composition,quantity,and function of intrahepatic immune cells,resulting in different infection outcomes.