Expression And Antiviral Function Of Toll-like Receptor 7 In Chronic Hepatitis B Patients And Its Correlation With Peg-IFN-α-2a Therapy

BackgroundChronic hepatitis B virus(HBV)infection remains one of the most common and serious public health problems around the world.And it is still have far to go to look for noval therapeutic targets to cure this disease.Current knowledge suggested that the impaired immune system,including the imblalance of immune function which mediated by toll-like receptor(TLRs),is a major reason for HBV latency,chronicity and reactivation.TLRs have been known as a bridge between naive and adaptive immunity,for their recognitions of multiple pathogens and induction of host immunity.It had been demonstrated that the application of TLR7 ligand(TLR7-L)could efficiently suppresses HBV replication in mouse,woodchuck and chimpanzee models.Moreover,Our group also found that the supernatant of PBMC from chronic hepatitis B(CHB)patients with TLR7-L stimulation could inhibit HBV replication in HepG2.2.15 cells.These findings suggested that TLR7 might be a promising condidate for developing new therapeutics for the treatment of chronic HBV infection.AimsTo investigate the expression of TLR7 in different stages during chronic hepatitis B infection and its regulation by antiviral therapy,and to further explore its potential antiviral mechanism in CHB patients.Materials and Methods1.Subjects1.1 Cross-sectional studyA total of 106 patients with chronic HBV infection(34 immune tolerance[IT],39 immune active[IA]and 33 inactive carriers[IC])and 31 health controls(HCs)were included.1.2 Longitudinal study25 HBeAg+CHB patients treated with Peg-IFN-a-2a were divided into off-treatment sustained virological response(SVR,HBeAg seroconversion and HBV DNA<1000 copies/mL at 24 weeks after discontinuation)group and non-off-treatment sustained virological response(NSVR)group.2.Animal model6-8 weeks old male C57BL/6j mice were hydrodynamic injected with pAAV HBV 1.2 and/or pKCMvint.IFN-a-2a plasmid,combined with or without TLR7-L to monitor the expression of HBsAg and HBeAg regularly and to detect intrahepatic and spleen specific or non-specific CD8+ T cell responses.3.Experimental methodPeripheral blood mononuclear cells(PBMCs)were separated by gradient centrifugation.The expression of TLR7 and other target genes were determined by realtime RT-PCR.The target cell subsets(CD19+ B cells,etc.)were prepared by Fluorescent cell sorting or magnetic bead sorting.Cytokine secretion,proliferation,activation and antibody secretion of target cells were determined by flow cytometry,enzyme-linked immunosorbent assay,and enzyme-linked immunospot assay,respectively.Results1.Cross-sectional studyDuring chronic HBV infection,the expression of TLR7 in IT group and IC group was significantly lower than that in HC group,while the expression of TLR7 in IA group was significantly restored.And TLR7 mRNA levels in IA phase was positively correlated with ALT levels2.Longitudinal studyThe expression of TLR7 in PBMCs of 25 patients with Peg-IFN-α-2a therapy was increased steadily during the treatment period.And there was a statistically significant difference of TLR7 epxression at week 24 between SVR group and NSVR group,suggesting that patients with a stable high TLR7 mRNA levels at week 24 were easier to undergo a sustained virological response after discontinuation of treatment.3.Functional expreiments3.1 The antiviral effect of TLR7 in vitroTLR7-L stimulation could induce a high amount of IFN-α secretion in PBMCs,while those from IA patients were significantly higher than from HCs.Exposeure of HepG2.2.15 cells to the PBMC supernatants by TLR7-L stimulation could strongly inhibited the HBV replication and antigen expression.At the same time,IFN-a stimulation could increase the expression of TLR7 in PBMCs in turn,suggesting that there was a positive feedback between TLR7 and IFN-α.3.2 Effects of TLR7 on the fucntion of B cellsTLR7 was highly expressed in B cells,and the expression of TLR7 on B cells was significantly increased after IFN-α stimulation.TLR7-L could work synergistically with IFN-α to significantly enhance the proliferation,activation,cytokines expression and antibody secretion of B cells.3.3 The antiviral effect of TLR7 in vivoTLR7-L was able to work synergistically with IFN-α to significantly increase the frequency of CD8+ T cells in intrahepatic lymphocytes and spleen lymphocytes.Moreover,the combined stimulation of TLR7-L and IFN-α could promote the recruitment of specific CD8+T cells from spleen to the target organ liver.At the same time,the IFN-γ and TNF-α secretion of specific and non-specific CD8+T cell in the intrahepatic and spleen were also largely increased after TLR7-L and IFN-αstimulation.ConclusionsTLR7 could inhibit the replication of HBV by inducing IFN-α secretion,while its expression and antiviral activity were up-regulated in IA patients.In addition,TLR7 expression was upregulated by Peg-IFN-α-2a treatment,and high TLR7 expression at 24 weeks may be associated with sustained virological response after discontinuation.And TLR7-L can work synergically with IFN-α to enhance the B cells function in vitro and the CD8+T cells responses in mice with chronic HBV infection.Our study implied that sequential therapy strategy with Peg-IFN-α-2a and TLR7-L may have a great potential for clinical application in CHB patients.