The Mechanism Of Activating STING Signaling Pathway To Induce Anti-HBV Immune Response

Background and aims:Hepatitis B virus(HBV)infection is a public health problem which seriously threatens human health.The host cannot produce enough immune response to clear HBV is related with the formation of chronic HBV infection.Current existing treatment plan for HBV infection can only inhibit HBV replication,which hardly achieve the functional cure of HBV infection.On the basis of the existing direct antiviral drugs,activating host immune system and recovering the immune response against hepatitis B virus is a hot topic in recent years.Pattern recognition receptors in innate immune response can recognize pathogen related molecular patterns,after being activated,they can induce the expression of type I interferon,inflammatory cytokines and interferon stimulating genes.Pattern recognition receptors mainly include DNA recognition receptors,toll like receptors,RIG-I receptors,et al.Cyclic GMP-AMP synthase(c GAS)is a DNA recognition receptor in cytoplasm,after recognizing the double-stranded DNA structure of pathogen or tumor,c GAS synthesized 2'3'-c GAMP,activating downstream key proteinstimulator of interferon genes(STING),and then induce the secretion of type I interferon(IFN)and inflammatory cytokine to eliminate pathogen or tumor cells.Recent research has shown that HBV escape the recognition of c GAS-STING signaling pathway may be an important reason for chronic HBV infection,however,there is little research has been performed on this protein in the field of hepatitis B treatment.Carry out relevant clinical research and explore whether STING agonist can effectively inhibit HBV replication and its immunological mechanism is very necessary.Methods:In this study,we collected the clinical samples of HBV infected patients in different immune stages,including peripheral blood and liver tissue.The peripheral blood samples were from 59 chronic HBV patients(CHB)and 13 socially recruited healthy volunteers who were treated in First hospital of Jilin University from March2019 to December 2019.Plasma and peripheral blood mononuclear cells were isolated from peripheral blood for follow-up experiments.Liver biopsy specimens were obtained from the pathological specimen center of Jilin University,including liver biopsy specimens from 19 chronic hepatitis B infected patients and 6 normal liver tissue specimens from patients with hepatic hemangioma.We successively measured the liver function and HBV virological markers in peripheral blood,and detected the expression of IFN-?,TNF-?,IP-10 by Luminex technique;In peripheral blood monocytes,the m RNA expression of STING and other pattern recognition receptor(PRR)were detected by real-time reverse transcription polymerase chain reaction(RT-PCR);For tissue samples,immunohistochemical staining methods were used to detect STING expression and macrophage polarization type in liver tissue.The effect of STING agonist on macrophage function was further detected by cytological experiment in vitro.Here,human monocytic leukemia cell line(THP-1)was induced differentiation into macrophages,then they were stimulated by different concentrations of STING agonist.Enzyme-linked immunosorbent assay(ELISA)was performed to detect the expression of cytokines,including,IFN-?,IL-6 et al.The liver tissue of HBV infected patient were collected and perfused in vitro,human primary hepatocytes and intrahepatic macrophages carrying HBV virus were isolated and cultured in vitro,we further explore the antiviral effect of STING agonist in human primary cells.In animal experiment,the mouse model of HBV infection was built by injecting r AAV8-HBV 1.3 vector via the tail vein.Blood was taken through the inner canthus vein regularly after HBV vector injection,the HBV DNA in the serum was detected by real-time PCR,HBs Ag and HBe Ag were detected by chemiluminescence microparticle immunoassay to verify whether the AAV-HBV model was successfully build.During administration period,The weight change and transaminase level of mice were monitored,and level of IFN and other cytokines in peripheral blood were detected by ELISA and flow cytometry.The changes of HBV DNA,HBs Ag and HBe Ag in peripheral blood were detected by q PCR and electrochemiluminescence.Liver tissue of mice were collected,for detecting the gene expression of interferon and inflammatory factors in liver and intrahepatic HBV DNA,additional liver tissue was taken for pathological correlation analysis,and fresh liver tissue was frozen for subsequent transcriptome sequencing.The length and weight of mouse spleen were measured,the spleen index was calculated,and the lymphocyte typing of spleen was detected by flow cytometry.Further,in the HBV infected mouse model,we explored the immunological mechanism of anti-HBV effect of STING agonist.We explored the targeting cells that mediate the antiviral effect of STING agonist.Macrophages,T cells were depleted in vivo by using chlorophosphonate liposomes and injection invivo T cell antibodies,the interferon signaling pathway was blocked by invivo antibody,thus,we evaluate the effects of different immune cells and interferon signaling pathway on the antiviral effect of STING agonist.By transcriptome sequencing,we detect the changes of m RNA expression level in mouse liver tissue after STING agonist administration,finding the pivotal differentially expressed genes,and clarify the signaling pathway enriched by differentially expressed genes through functional enrichment analysis.Result:According to the level of HBV virological markers and alanine aminotransferase level,the patients had no anti-viral treatment before were divided into patients in immune tolerance phase,immune activation phase,inactive low-level replication phase,healthy volunteers was set as control.The results showed that the m RNA expression of STING,Toll like receptor 8 in immune active phase was significantly higher than that in immune tolerance phase(P<0.05).Cytokine level,including IP-10 and TNF-? also increased significantly in the immune activation phase(P<0.05),suggesting the activation of immune system at this stage.In the liver tissue of patients with chronic HBV infection,we detected that the expression of STING in immune active phase was higher than that in the inactive phase and healthy control group(P < 0.05).There was no significant difference between patients in immune tolerance phase and normal people.In vitro experiment,THP-1 cells were induced into macrophages,after stimulating by STING agonist,the level of IFN-? and IL-6 were increased significantly in the supernatant of culture medium,which was dose-dependent with the concentration of STING agonist.Further,we found in in vitro culture model of primary human hepatocytes and intrahepatic macrophages of HBV infected patients,the participation of intrahepatic macrophages can enhance the inhibition of STING agonist on HBV replication.In animal experiments,we detected that STING agonist can activate the immune response in AAV-HBV infected mice.Compared with mice given placebo,in mice treated with STING agonist,we detected the level of IFN-? and IL-6 in serum,the interferon and inflammatory factor gene expression in liver,and spleen index and spleen volume were all increased significantly(P < 0.05).The anti-HBV efficacy of STING agonist was further evaluated in AAV-HBV infected model,the result showed that STING agonist could reduce the levels of HBV DNA,HBs Ag and HBe Ag in serum,especially in HBV DNA,which was correlated with the dose of STING agonist.In mice liver tissue,the decrease of HBs Ag and intrahepatic HBV DNA level in STING agonist treatment group could also be detected.We also evaluated the effect of STING agonist combined with entecavir,compared with STING agonist monotherapy,the decrease of HBV DNA in the combined group was more obvious.In the exploration of immune mechanism,we evaluated the anti-HBV effect of STING agonist in the immune cell defective animal model.The results showed that,the intrahepatic macrophages and T cells were all involved in the anti-HBV effect of STING agonist,while,in terms of the degree of inhibition,macrophages are more critical to the anti-HBV effect of STING agonist.After blocking interferon receptor,most of the up-regulated interferon and inflammatory factor genes induced by STING agonist were inhibited,also the inhibit effect on HBV replication was weakened,which confirm the key role of interferon signal pathway in mediating the immune activity of STING agonist.Transcriptome sequencing of liver tissue in STING agonist cohort and control cohort were carried out on the liver samples of experimental animals,a total of856 differentially expressed genes(DE m RNAs)were screened out,including 665 upregulated genes and 191 down-regulated genes.By GO analysis,we found that differentially expressed genes in biological processes were mainly enriched in signals,such as transduction and cell transformation;in terms of cellular components,they were enriched in cellular components such as intercellular connections and macromolecular complexes;in terms of molecular function,they were mainly concentrated in binding,catalytic activity.Further Go enrichment analysis showed that the differentially expressed genes were enriched in chemokine receptor activity,2'5 'oligoadenylate synthase(OAS)activation,monocyte chemotaxis and antigen presentation.Enrichment analysis of KEGG pathway showed that the differentially expressed genes were enriched in immune related signal pathways such as phagocytosis,extracellular matrix receptor recognition,intercellular adhesion and antigen presentation.Conclusion:As a DNA recognition receptor,in innate immune response system,STING signaling pathway plays an important role in recognizing pathogen DNA.In this study,we revealed the correlation between STING expression and inflammatory levels in different immune phases of chronic HBV infected patients,detected the polarization state of intrahepatic macrophages in chronic HBV infected patients,and preliminarily evaluated the anti-HBV effect of STING agonist in human HBV infected primary hepatocytes,which provided evidence for exploring the clinical significance of STING signaling pathway in the pathophysiological mechanism of chronic HBV infection.By building r AAV8/1.3 HBV chronic HBV infection mouse model,we detected STING agonist could effectively induce the immune response and anti-HBV response.In this process,intrahepatic macrophage and T lymphocyte mediate the antiviral effect of STING agonist,which accompanied by the up-regulation of multiple immune related genes in the liver and the activation of immune pathways.This study provides more theoretical evidence for the research and clinical application of STING agonist in the field of HBV treatment in the future.