The Effect And Mechanism Of Thrombopoietin Receptor Agonist Eltrombopag In Type ? Interferon Pathway

BackgroundThrombocytopenia is one of the common complications of chronic viral hepatitis.Especially,the incidences of thrombocytopenia in chronic hepatitis C(CHC)and chronic hepatitis B(CHB)are higher clinically.The patients with chronic viral hepatitis not yet progressing to cirrhosis usually manifest mild to moderate thrombocytopenia(platelet counts of 50-150×109/L).The patients with severe thrombocytopenia(platelet counts of less than 50×109/L)may manifest mucocutaneous bleeding,even visceral bleeding.Thrombocytopenia impedes anti-viral treatments as well as is relevant to poor prognosis.Immune thrombocytopenia(ITP)can be divided into primary and secondary ITP.The therapeutic methods of primary and secondary ITP are similar in a number of situations.But the treatment of ITP secondary to underlying diseases such as hepatitis C virus(HCV)infection and hepatitis B virus(HBV)infection should focus on the infection itself.However,severe thrombocytopenia can impede or postpone the use of anti-viral drugs.Therefore,the therapeutic principle of thrombocytopenia associated with chronic hepatitis viral infection involves mainly two aspects:anti-viral treatments based on etiology and treatments on secondary thrombocytopenia.Type ? interferon(IFN)is an important effector molecule of anti-viral immune response.Binding with type ? interferon receptor(IFNAR)activates downstream Janus kinase(JAK)-Signal transducer and activator of transcription(STAT)signaling pathway.Then IFN-stimulated genes(ISGs)are induced to exert anti-viral function.Thereby,Pegylated interferon-alpha(Peg-IFN?)is recommended in guidelines as first-line anti-viral drugs extensively used to treat chronic viral hepatitis(CHC and CHB).In regard to patients with chronic viral hepatitis and thrombocytopenia not yet progressing to cirrhosis or compensated cirrhosis,it is currently inclined to proceed anti-viral therapy based on referring to consensus guidelines for the management of ITP.However,owing to the probability of viral activation,thereby aggravating symptoms through the administration of steroids,there are certain limitations in clinical applications.In view of this,it is urgent to find safe and effective new therapeutic drugs to increase platelet counts of chronic viral hepatitis with thrombocytopenia patients.Eltrombopag is the thrombopoietin receptor(TPO-R)agonist and has become the second-line drug of ITP.What's more,eltrombopag has been applied to treat CHC patients with thrombocytopenia.Eltrombopag facilitates CHC patients with low platelet counts can initiate and maintain standard treatments for liver disease based on IFN.The mechanism of eltrombopag is similar to that of endogenous thrombopoietin(TPO),stimulating platelet production through binding to the TPO receptor(TPO-R).In addition,eltrombopag has another critical function of iron chelation.Eltrombopag can influence the functions of hematopoietic stem cells by decreasing intracellular iron.Monocytes can differentiate into macrophages and dendritic cells,which are involved in antigen presentations of hepatitis virus and respond to the immune therapies of chronic viral hepatitis.However,it is unclear whether eltrombopag affects type ?interferon immune response as the potential therapeutic drug of thrombocytopenia associated with chronic hepatitis viral infection.There is no relevant research yet.This study has reference value for the systematic understanding of the role of eltrombopag in the treatment of chronic viral hepatitis with thrombocytopenia.Objective1.Explore the effects of eltrombopag on the IFN-?-induced anti-viral immune response.2.Uncover the molecular mechanism of eltrombopag in regulating type ? interferon immune responses in monocytes.Methods and results1.Eltrombopag inhibited type ? interferon signaling pathway independently of TPO-R.1.1 Eltrombopag differently regulated the activation of ISGs in human myeloid leukemia mononuclear cells and human monocytes.Collect peripheral blood from healthy volunteers,and Ficoll density gradient centrifugation method was used to separate peripheral blood mononuclear cells(PBMCs).Then CD 14 positive monocytes were sorted from PBMCs using CD 14 immunomagnetic beads.The purity of more than 95%was measured by flow cytometry.Cell Counting Kit-8(CCK-8)was used to access cell viability following THP-1 cells and CD 14 positive monocytes treated with different concentrations of eltrombopag.The CCK-8 analysis revealed that concentrations below 10 ?M of eltrombopag did not affect the viability of two types of cells.In addition,the blood concentration of eltrombopag in multiple clinical trials is higher than 10 ?M.Thus,eltrombopag at the concentration of 10 ?M is qualified for pharmacological and pathological conditions.THP-1 cells treated with eltrombopag in vitro were collected,then total RNA was extracted from THP-1 cells,and cDNA was obtained by reverse transcription.Quantitative Real-time PCR(qPCR)was performed to detect the expression levels of ISGs.qPCR results showed an increase in secreted factors(including CXCL10,IFIT1,IFIT2,ISG15,and MX2)in eltrombopag-pretreated THP-1 cells primarily upon IFN-?stimulation.After treatment in vitro with eltrombopag,human monocytes were collected,and ISGs mRNA expression levels were detected by qPCR.The results showed that eltrombopag reduced IFN-?-induced ISGs mRNA expressions in human monocytes.1.2 Eltrombopag regulated the activation of ISGs which was relevant to TPO-R.In view of the classic function of eltrombopag as the TPO-R agonist,to explore the mechanisms that contribute to the differential regulation of ISGs between THP-1 cells and human monocytes,the total proteins of THP-1 cells and human monocytes were extracted respectively,and the expression levels of TPO-R were detected by western blotting.As a result,it was found that the expression of TPO-R was significantly different between the two kinds of cells.The transfection complex containing small interfering RNAs(siRNAs)and transfection reagents was added to THP-1 cells.After 48 hours,the cells were collected,and the interference efficiency was detected by western blotting.TPOR gene was down-regulated by siRNA transfection in THP-1 cells.qPCR results showed that TPOR knockdown significantly suppressed the enhancement effect of eltrombopag treatment on IFN-?-induced ISGs expression.The above results indicated that the different regulations of the activation levels of ISGs in THP-1 cells and human monocytes might be related to TPO-R expression.1.3.Eltrombopag inhibited the production of ISGs induced by IFN-?independent of TPO-R.Because eltrombopag cannot activate murine TPO-R,mice become an ideal model for studying the TPO-R-independent effects of eltrombopag.Bone marrow cells from femurs and tibias of wild-type C57BL/6J mice were harvested,and bone marrow mononuclear cells(BM-MNCs)were isolated using a murine bone marrow mononuclear cell separation kit.BM-MNCs will be adherent within 2-4 hours after cell culture.BM-MNCs were treated with respectively different concentrations or different times of eltrombopag.qPCR results showed that EP inhibited ISGs expression in murine BM-MNCs in the dose and time-dependent manners.After BM-MNCs were treated with eltrombopag in vitro,western blotting showed that eltrombopag inhibited IFN-?-induced downstream STAT1 phosphorylation.Different from eltrombopag,both recombinant human thrombopoietin(rhTPO)and another thrombopoietin receptor agonist(TPO-RA)romiplostim(Romi)can bind to murine TPO-R.qPCR and western blotting showed that rhTPO and Romi had no significant effect on IFN-? downstream STAT1 phosphorylation and ISGs expression in murine BM-MNCs.These results suggested that eltrombopag suppressed type ? interferon signaling pathway in a TPO-R-independent fashion.2.Eltrombopag inhibited type ? interferon signaling pathway through chelating intracellular iron.2.1 Iron metabolism was found to play a vital role in this process via transcriptome sequencing of ex vivo eltrombopag-treated monocytes from CHB with thrombocytopenia patients.Peripheral blood of CHB with thrombocytopenia patients was collected.Then CD 14 positive monocytes were sorted by immunomagnetic beads and treated with eltrombopag in vitro.Total RNA was extracted for transcriptome sequencing(RNA-seq).Sequencing results were analyzed by R and RStudio software.We screened out 91 differentially expressed genes(DEGs)with a corrected p-value of less than 0.05.Of these,the novel iron metabolism-related gene BOLA2 was found to be downregulated in eltrombopag group.The role of the metabolism-related gene in eltrombopag function can further be verified using siRNA to downregulate the Bola2 gene in murine BM-MNCs.And qPCR results showed that Bola2 downregulation inhibited IFN-?-induced ISGs expression.These data indicated that eltrombopag regulated iron metabolism-related mechanisms to inhibit the IFN-? pathway.2.2 Eltrombopag inhibited ISGs expression through chelating iron in monocytes.Deferoxamine(DFO)is a classic iron chelator.First,CCK-8 was used to access cell viability following stimulation of different concentrations of DFO.The CCK-8 analysis revealed that concentrations below 20 ?M of DFO did not affect the viability.Then qPCR and western blotting showed that DFO treatment reduced STAT1 phosphorylation and downregulation of ISGs,consistent with eltrombopag treatment.Eltrombopag-treated murine BM-MNCs exhibited a significant decrease in ISGs mRNA expressions followed by IFNA2 stimulation,partly restored by the co-incubation of eltrombopag with ferrous sulfate(FeSO4).Intracellular labile iron pool(LIP)alterations were evaluated using an iron-binding compound Calcein AM.After incubation with Calcein AM,the cells were then treated with eltrombopag.Intracellular calcein means fluorescence intensity(MFI)values were obtained by flow cytometry and analyzed by FlowJo.Flow cytometry analysis showed an increase in calcein-AM fluorescence in cells treated with eltrombopag,which indicated the reduction of free intracellular iron.Collectively,our results suggested that eltrombopag diminished ISGs production via iron chelation in monocytes.3.Eltrombopag inhibited type ? interferon signaling pathway via regulating iron-mediated reactive oxygen species production.3.1 Eltrombopag inhibited intracellular reactive oxygen species production.Iron in the oxidative state participates in reactive oxygen species(ROS)production through the Fenton reaction.Thus,we assessed intracellular ROS levels in eltrombopag-pretreated human monocytes.ROS level was measured using 2,7-dichlorofluorescein diacetate(DCFDA).After incubation with DCFDA,the cells were then treated with eltrombopag,and the corresponding increase in fluorescence was recorded continuously on a microplate reader.The results showed that eltrombopag progressively decreased endogenous and abundant exogenous ROS levels compared to controls.3.2 Eltrombopag reduced ROS generation to inhibit the JAK activation and ISGs expression.To determine whether ROS was involved in eltrombopag-mediated IFN-?downstream suppression in monocytes,we incubated murine BM-MNCs with hydrogen peroxide(H2O2)during eltrombopag treatment.qPCR results showed that co-exposure of eltrombopag and H2O2 resulted in reverse IFN-?-induced ISGs expressions,compared with eltrombopag treatment alone.Western blotting showed that eltrombopag treatment significantly suppressed the phosphorylation of JAK1 and STAT1,which could be affected by ROS.These results suggested that eltrombopag decreased ROS production via chelating intracellular free iron,accordingly inhibited the IFN-?-induced phosphorylation of JAK and downstream ISGs expressions.ConclusionThis research verified that the eltrombopag's iron-chelating character led to a decrease in ROS generation via reduced free iron.Thereby phosphorylation of JAK1 and STAT1 and ISGs expressions induced by IFN-? were suppressed.In conclusion,eltrombopag inhibited the type ? interferon antiviral signaling pathway.This research is valuable to systematically understand the role of eltrombopag in treating chronic viral hepatitis with thrombocytopenia,and more attention should be paid in this area.