Study On The Mechanism Of Eitrombopag In Chronic Hepatitis B-associated Thrombocytopen

Part Ⅰ:Transcriptome analysis in the chronic hepatitis B-associated thrombocytopenia with eltrombopag treatmentBackground:Secondary thrombocytopenia is common in the clinic,mainly manifested by bleeding,including epistaxis,gingival,oral mucosa,and eye binding membrane bleeding,spontaneous skin petechia,ecchymosis or purple spot.It is often secondary to autoimmune diseases such as systemic lupus erythematosus and infectious diseases including human immunodeficiency virus infection and chronic hepatitis virus infection.Chronic hepatitis B(CHB)is a chronic liver disease caused by persistent hepatitis B virus(HBV)infection,which affects 296 million people in the world.Meanwhile,it has been reported 70 million patients are infected by HBV in China.The prevalence of thrombocytopenia is 17.7%in patients with chronic hepatitis B virus and 10.6%in carriers.The degree of thrombocytopenia has been shown to be a useful marker for early prognosis in patients with cirrhosis.Pegylated-interferon(PEG-IFN)is an important therapeutic method for chronic hepatitis.Thrombocytopenia associated with chronic liver disease may affect the antiviral effect of PEG-IFN.Therefore,effective management of platelet counts in hepatitis patients before and during antiviral therapy is of certain significance.There are no guidelines for the treatment of thrombocytopenia related to chronic hepatitis currently.The therapy referred to primary immune thrombocytopenia.About 50%of patients respond to the hormone,and a transient application of immunoglobulin to suppress HBV immune response can temporarily increase platelets.Splenectomy may be considered for patients with chronic hepatitis-associated thrombocytopenia who do not respond well to traditional therapies,but there may be risks of infection,portal vein thrombosis,etc.In recent years,a large number of studies have gradually confirmed the key regulatory role of thrombopoietin(TPO)in the process of thrombocytopenia.Therefore,the clinical effects and immune mechanisms of TPO and its receptor agonists in the treatment of thrombocytopenia are also being explored.Eltrombopag(EP)is an oral,small-molecule,non-peptide TPO receptor agonist.It binds to the transmembrane region of the TPO receptor and activates intracellular signal transduction pathways.This induces the proliferation and differentiation of megakaryocyte precursors and megakaryocytes,thereby increasing platelet counts.Studies have shown that eltrombopag increases platelet counts in patients of chronic hepatitis C(CHC),advanced fibrosis,and cirrhosis with thrombocytopenia,enabling patients who cannot be treated due to low platelet counts to initiate and maintain antiviral therapy,and thereby significantly improving sustained virological response(SVR).No clinical trials have been published for the treatment of CHB-associated thrombocytopenia with eltrombopag.A study published in 2012 reported that eltrombopag reduced the demand for platelet transfusion in patients with chronic liver disease undergoing selective invasive surgery.Therefore,it is expected that eltrombopag can achieve better efficacy in CHB-associated thrombocytopenia.Our team is recruiting patients with CHB-associated thrombocytopenia to evaluate the safety and effectiveness of eltrombopag,and we have got preliminary positive clinical data.In recent years,deep sequencing has revolutionized biological and medical research.Transcriptome sequencing(RNA-seq)is a second-generation sequencing technology that can analyze the entire transcriptome,thus revealing changes in the entire signal network and helping to identify novel significant genes that cannot be predicted in advance.It is currently the preferred method for studying gene expression and identifying new RNA types in biology and medicine.In this study,transcriptome sequencing was conducted on peripheral blood mononuclear cells(PBMCs)of patients with CHB-associated thrombocytopenia before and after eltrombopag treatment to discover the key genes and reveal the important molecular biological mechanism of eltrombopag treatment.Finally,this study can provide a new perspective for platelet elevation and antiviral therapy in patients with CHB-associated thrombocytopenia.Objective:To explore the transcriptome changes of PBMCs in patients with chronic hepatitis Bassociated thrombocytopenia after treatment with eltrombopag.Methods and results:1.Patients with chronic hepatitis B-associated thrombocytopenia,whose HBsAg and/or HBV DNA positive for at least six months and platelet was lower than 100*109/L,were recruited and given eltrombopag treatment for 6 weeks.Peripheral blood(10ml)was collected before and after treatment,PBMCs were extracted and RNA-Seq was performed.Through the library construction and sequencing,data quality control,and comparison reference gene for gene expression,genes with |log2FoldChange|≥1,p-value<0.05 was selected as differential expression genes.Finally,782 up-regulated genes and 206 down-regulated genes were screened,which were presented in the form of cluster analysis heat map and volcano map.2.GO enrichment analysis for differential expression genes was further conducted,including three aspects:biological process(BP),cellular component(CC)and molecular function(MF).The results showed that the enrichment of biological processes was mainly related to the molecular response of bacterial infection and the regulation of inflammatory response.In terms of cell components,it is mainly involved in the formation of plasma membrane and blood droplets.For molecular function,it is mainly concentrated in MAPK tyrosine/serine/threonine phosphatase activity,cytokine activity,and G protein-coupled receptor binding.3.KEGG enrichment analysis was used to search for the signal pathway of significant enrichment.It was considered that KEGG term with a p-value less than 0.05 was significantly enriched through differentially expressed genes.The results showed that the differential expression genes were enriched in the IL-17 signaling pathway,MAPK signaling pathway,rheumatoid arthritis,and influenza A virus-related pathways.4.In order to observe subtle gene expression changes,Gene Set Enrichment Analysis(GSEA)was performed.This is an analytical method that does not need to specify a clear differential gene threshold,uses a pre-defined gene set,ranks the genes according to the degree of differential expression in the two types of samples,and then tests whether the predefined gene set is enriched at the top or bottom of the sequencing table.The results showed that the gene sets related to antigen binding function,lymphocyte-mediated immune response,leukocyte-mediated immune response and B cell-mediated immune response was more enriched.And lymphocyte-mediated immunity intersects with many other enriched gene sets.5.Total RNA was extracted from peripheral blood mononuclear cells before and after eltrombopag treatment,and cDNA was obtained by reverse transcription.Through primer design and real-time quantitative PCR calculation,CD69,DUSP8,RGS1,DUSP4,and other genes were verified to be consistent with the results of RNA-Seq.Conclusion:Transcriptome analysis showed that 988 genes changed significantly after eltrombopag treatment,including 782 up-regulated genes and 206 down-regulated genes.Real-time quantitative PCR verified the analysis results.GO analysis of differential expression genes showed that DEGs function is related to the molecular response of bacterial infection,regulation of inflammatory response,cytokine activity,and G protein-coupled receptor binding.KEGG pathway enrichment analysis further explored the pathway annotation of differential expression genes that were associated with the IL-17 signaling pathway,MAPK signaling pathway,rheumatoid arthritis,and influenza A virus.In addition,GSEA analysis revealed higher enrichment of gene sets related to humoral and cellular immunity,and lymphocyte-mediated immune response interacted with many other enriched gene sets.Part Ⅱ:Eltrombopag plays an antiviral role by enhancing the function of CD8+CXCR5+T cellsBackground:More than 296 million people around the world are chronically infected with the hepatitis B virus,and China is one of the world’s leading countries in the incidence of liver disease,which brings a huge social and economic burden to China.If chronic hepatitis B virus infection is not effectively controlled,it will progress to cirrhosis and even hepatocellular carcinoma,which seriously threatens the life of patients.Factors that promote the development of hepatocellular carcinoma include long-term HBV infection and high levels of HBV replication.The higher the level of hepatitis B virus in the serum,the more severe liver damage and the more likely the disease is to progress.Therefore,effective control of viral load in patients with chronic hepatitis B is an important measurement to prevent progression to hepatocellular carcinoma.The goal of hepatitis B virus therapy is to achieve deep virological inhibition.Functional cure is defined as continuous inability to detect hepatitis B surface antigen and hepatitis B virus DNA without antiviral therapy(AVT),which means that hepatitis B virus replication can be completely inhibited in the liver even at the subclinical level.At present,the main antiviral treatments for chronic hepatitis B are pegylated interferon therapy and nucleotide reverse transcriptase inhibitors(NRTI)therapy.According to statistics,pegylated interferon therapy may achieve functional cure in 10%of patients,but it may cause bone marrow suppression and autoimmune diseases.For NRTI therapy,because it targets the reverse transcription process to inhibit HBV DNA replication and does not eliminate the virus completely,only a small number of patients can achieve functional cure,and severe erythema and hepatic decompensation may occur when stopping NRTI therapy.Therefore,functional cure of chronic hepatitis B patients through effective therapeutic means is the current needs of chronic hepatitis B patients.Exhaustion of T cells is considered to be an important cause of hepatitis B virus infection.Sufficient enhancement of HBV-specific immunity through immunotherapy is challenging due to extremely low HBV-specific T cells level and weak T-cell responses,which are associated with immune failure,immune dysregulation,and immunosuppressive pathways.Recent studies have found that CD8+CXCR5-T cells,a novel T-cell subgroup,are present in the peripheral blood of patients with chronic hepatitis B,which has a similar phenotype to follicular helper T cells.Co-culture of CD8+CXCR5+T cells and B cells can increase the differentiation of B cells into plasma cells,and enhance the antiviral effect.Clinical evidence also shows that in CHB patients,the proportion of CD8+CXCR5+T cells is higher in patients with low HBsAg,low HBV DNA level,HBeAg negative,and high hepatic fibrosis score,and is significantly correlated with the decline of HBsAg and HBV DNA.Eltrombopag(EP),a seconed-line therapy of primary ITP,has been reported to inhibit the proliferation of leukemia cell lines and human cytomegalovirus(HCMV)replication in human fibroblasts and adult mesenchymal stem cells.However,it is unknown whether eltrombopag has inhibition effects to hepatitis B virus.This part of the study explores antiviral immune effects of eltrombopag in CHB-associated thrombocytopenia besides promoting platelet production,which provides new perspective for platelet promotion and antiviral therapy in patients with CHB-associated thrombocytopenia and has guiding significance for clinical drug combination in such patients.Objective:To explore the antiviral immune regulation of eltrombopag in patients with chronic hepatitis B-associated thrombocytopenia.Methods and Results:1.Firstly,we detected the protein expression level of CD69.Since CD69 is a recognized marker on the surface of T cells,we used flow cytometry to detect CD69 levels on T cell surfaces.The results showed that CD69 on the surface of CD3+,CD4+and CD8+T cells increased significantly after eltrombopag treatment.When T cell subsets were focused,the expression ratio of CD69 on the surface of CD8+T cells was significantly higher than that of CD4+T cells both before and after the eltrombopag treatment.Therefore,we believed that CD69 is mainly expressed by CD8+T cells and plays a major immune role.2.Patients were divided into two groups:chronic hepatitis B-associated thrombocytopenia group and chronic hepatitis B without thrombocytopenia group according to whether they were complicated with thrombocytopenia(PLT<100*109/L)or not.It was found that CD69 expression in patients with chronic hepatitis B-associated thrombocytopenia was significantly elevated after eltrombopag treatment,while chronic hepatitis patients without thrombocytopenia did not,suggesting that the effector function of eltrombopag was specific.3.CD8+CXCR5+T cells and CD8+CXCR5-T cells were distinguished by surface antibody staining,and the proportion changes of the two cell subsets and the expression of CD69 were counted respectively before and after eltrombopag treatment.The results showed that both the proportion of CD8+CXCR5+T cells and CD8+CXCR5-T cells were no significant change.At the same time,CD69 activation on the surface of CD8+CXCR5+T cells were stronger than that on CD8+CXCR5-T cells.4.We used hepatitis B antigen peptide to stimulate peripheral blood mononuclear cells of patients with CHB-associated thrombocytopenia and measured the degree of granulation and cytotoxic activity of activated T cells through CD 107a expression.The results showed that the expression of CD 107a on the surface of CD8+CXCR5+T cells was significantly increased in the eltrombopag treatment group compared with the control group after stimulation with hepatitis B antigen peptide,while the expression of CD 107a on the surface of CD8+CXCR5-T cells was not significantly changed.5.Hepatitis B antigen peptide was used to stimulate peripheral blood mononuclear cells of patients,and the function of activated T cells was evaluated by detecting intracellular factors.Results showed that IFN-γ and TNF-α on CD8+CXCR5+T cells increased significantly with eltrombopag treatment after antigen stimulation,while there was no significant change in CD8+CXCR5-T cells.6.We used simple linear regression to analyze the relationship between the expression of CD107a,IFN-γ and TNF-α and the expression of CD69.CD107a and IFN-y were positively correlated with CD69 expression,while TNF-α had no correlation with CD69.7.Combined with clinical data,the enhancement effect of eltrombopag on CD8+T cells under different clinical characteristics was analyzed.It was found that the immune effects of eltrombopag was not related to the HBV DNA level,HBeAg status and AST level,but was related to the HBsAg and ALT level,and activation effects were more obvious in patients with low HBsAg and normal ALT.Conclusion:In this part,it was proved that CD8+effector T cells may be the main cell group activated by eltrombopag,and flow cytometry proved that CD8+CXCR5+T cells,the functional phenotype of exhausted T cell subgroup,participated in the response of eltrombopag.After the use of eltrombopag,the expression of CD69 on its surface was significantly increased,and in response to antigen stimulation,CD8+CXCR5+T cells showed significantly increased expressions of CD 107a,IFN-γ and TNF-α,indicating that eltrombopag may play an antiviral role in addition to improving platelet counts in the treatment of patients with CHB-associated thrombocytopenia.At the same time,it was proved that the response of eltrombopag had no correlation with the status of chronic hepatitis B infection,and was suitable for almost all patients with CHB-associated thrombocytopenia according to our clinical analysis.