| Primary immune thrombocytopenia(ITP)is an acquired autoimmune hemorrhagic disease.The main clinical manifestations are asymptomatic thrombocytopenia and haemorrhage involving the skin or mucous membranes,endometrium,visceral,or even intra-cranial in severe cases.And the risk of bleeding increases with age.ITP accounts for about 30%of clinical hemorrhagic diseases.The pathogenesis of ITP are complex:1.Abnormal humoral immunity.The body’s immune system is intolerant to autoantigens,and produces a variety of autoantibodies to combine with platelet membrane glycoprotein,which can increase platelet destruction by binding to FC γ receptor on the surface of macrophages;2.Abnormal cellular immunity.Excessive proliferation and activation of reactive T lymphocytes and B lymphocytes,imbalance of Th1/Th2,Th17/Treg subsets,secretion of a variety of inflammatory cytokines,resulting in immune disorders;3.Bone marrow megakaryocyte maturation disorder and increased destruction leading to thrombocytopenia.In addition to the pathogenesis mentioned above,there are still many causes of ITP.The first-line treatment of ITP is glucocorticoid and immunoglobulin.Many side effects of glucocorticoid seriously reduce the quality of life of patients,and about 15%-20%of patients are ineffective in the first-line treatment.Although there are a variety of second-line treatment drugs,such as CD20 monoclonal antibody,recombinant human thrombopoietin,recombinant human thrombopoietin receptor agonist,splenectomy and so on,about 1/3 of the patients still have poor response to the above treatment and become refractory ITP,These patients suffer from disease and economic pressure for a long time.Therefore,it is of great significance to further study the pathogenesis of ITP and search for new treatment methods.Ubiquitin-proteasome system is a non-lysosomal protein degradation pathway widely existing in eukaryotic cells.It plays an important role in protein transcription and translation,cell cycle regulation,signal transduction,apoptosis and immune response.The system consists of ubiquitin,ubiquitin activating enzyme,ubiquitin ligase,ubiquitin binding enzyme,deubiquitinase and 26S proteasome.26S proteasome is composed of 19S regulatory cap subunits and 20S protein subunit containing one 20S protein subunnit.All 20S particles consist of four stacked heptameric ring structures.The outer two rings are composed of a subunit and the inner two rings are composed of β subunit,forming a central proteolysis chamber.βring contains β1,β2 and β5 subunits,which are the catalytic active center of 20S proteasome.They are organized and expressed in all cells.When cells are induced by interferon(IFN)-y,tumor necrosis factor-a(TNF-α),inflammatory reaction and oxidative stress,β1,β2 and P5 subunits are replaced by LMP2(β1i)、LMP 10(β2i)and LMP7(β5i)to form an immunoproteasome.Compared with standard proteasome,immunoproteasome has higher chymotrypsin like activity and higher ability to process and present antigen.The evidence is accumulating that immunoproteasome is related to adaptive immune response and plays an important role in the pathogenesis of various inflammatory diseases.Selective inhibition of immunoproteasome subunit can relief the clinical symptoms of rheumatoid arthritis,multiple sclerosis,systemic lupus erythematosus,inflammatory bowel disease,diabetes and Hashimoto’s thyroiditis mouse models.Immunoproteasome inhibitors can prevent chronic antibody mediated allograft rejection in renal transplantation and promote long-term acceptance of mouse heart grafts.In another study,selective immunoproteasome subunit inhibitors can be used to target cells with high immunoproteasome expression,such as malignant tumor cells.Therefore,the clinical application of immunoproteasome inhibitors can reduce the side effects of proteasome inhibition and improve the treatment options in the future.The function of immunoproteasome was initially focused on antigen processing.It can efficiently produce antigen peptides that are beneficial to the binding of MHC-I molecules and are recognized by CD8+ T cells.With the deepening of research,more functions of immune proteasome have been reported,including regulating T cell survival,differentiation and proliferation,and affecting the production of related cytokines,such as IL-6,IFN-γ,TNF-α and IL-23.However,the role of immunoproteasome in ITP remains to be studied.The purpose of this study is to investigate the expression of immunoproteasome subunits in ITP patients.Through cell experiment in vitro and animal experiment in vivo to explore the role of immunosuppression in the pathogenesis of ITP,so as to further improve the studies of ITP and provide new treatment for ITP patients.Part Ⅰ Mechanism of immunoproteasome inhibitor on ITP patientsBackgroundImmunosuppression is a special type of proteasome,which is mainly appear in cells from hematopoietic stem cells.In addition,cells from non-hematopoietic cells can be induced to express the immunosuppression after being stimulated by IFN-y and TNF-α.LMP2,LMP10 and LMP7 are the hydrolytic subunits,which have caspase-like activity,trypsin-like activity and chymotrypsin-like activity respectively.Immunoproteasome can play a variety of functions by mediating proteolysis,such as regulating innate and adaptive immunity,effectively antagonizing oxidative stress,removing oxidative damaged proteins,and protecting cell function.With further research,it is found that appropriate production of immunoproteasome has a protective effect on the body.However,excessive amount of immunoproteasome may have a negative effect on cells,leading to the occurrence of some autoimmune diseases and tumor diseases.Studies have shown that immunoproteasome subunits in serum of patients with systemic lupus erythematosus(SLE),rheumatoid arthritis(RA),Sjogren’s syndrome(SS)and multiple sclerosis(MS)or animal models is increased,and the symptoms of these diseases can be improved by using subunit inhibitors or knocking out related gene.ITP is an immune-mediated thrombocytopenia disease.The study indicates that the expression of IFN-y in ITP patients increased,but the expression of the immune proteasome subunits in ITP patients is not clear.Previous researches have shown that immunoproteasomes not only deal with MHC restricted proteins,but also participate in regulating the differentiation of helper T cell.Inhibition of LMP7 or gene knockout can inhibit the differentiation of Thl and Th17,and enhance the production of regulatory T cells(Tregs).In addition,the selective inhibition of immunoproteasome has a negative regulatory effect on the production of proinflammatory cytokines such as IL-6,IFN-y,TNF-α,GM-CSF and IL-23.However,the mechanism of immunoproteasome in ITP remains to be studied.Objective1.Analyze the expression of LMP2 and LMP7 in peripheral blood mononuclear cells(PBMCs)of ITP patients and healthy controls.2.Analyze the effect and mechanism of immunoproteasome inhibitors on reversing the abnormal function of immune cells in ITP,and explore a new target of ITP treatment.Methods1.Thirty three ITP patients and thirty healthy controls were enrolled.Peripheral blood mononuclear cells(PBMCs)were isolated from 10 ml peripheral blood.The expression of LMP2 and LMP7 in total protein of ITP patients and healthy controls were detected by enzyme linked immunosorbent assay(ELISA).The mRNA expressions of LMP2,LMP7 and LMP10 in PBMCs of ITP patients and healthy controls were detected by real-time reverse transcription polymerase chain reaction(RT-PCR).2.CD 14+cells were isolated from PBMCs of ITP patients by positive selection with anti-CD 14 magnetic beads and then were treated with ML604440(LMP2 inhibitor),ONX-0914(LMP2 and LMP7 co-inhibitor)or DMSO respectively for 24 hours.The expression of CD 16(FcγRⅢ)and CD64(FcyRI)were detected by flow cytometry.3.Phagocytic function of macrophages:CD 14+cells were isolated from PBMC of ITP patients by positive selection with anti-CD14 micromagnetic beads.Macrophages treated with ONX-0914 were incubated with CMFDA(5-chloromethylfluorescein diacetate)labeled platelets which were coated anti-CD41 antibody for 1 hour.The average fluorescence intensity of platelets phagocytized by macrophages was measured by flow cytometry.4.PBMCs from ITP patients were cultured in vitro and were treated with ML604440,ONX-0914 or DMSO.The expression of CD69 and CD25 were detected by flow cytometry after 10 hours and 72 hours respectively.5.CD4+cells were isolated from PBMC of ITP patients by positive selection with anti-CD4 magnetic beads and were treated with ML604440,ONX-0914 or DMSO respectively for 72 hours.The expression of IFN-y and IL-17 were detected by flow cytometry.6.PBMCs from ITP patients were cultured in vitro and were treated with ML604440,ONX-0914 or DMSO for 72 hours.The protein expression of p-STAT1,STAT1,p-STAT3 and STAT3 was detected by Western blot.Results1.Compared with healthy controls,ITP patients had abnormal expression of immunoproteasome subunits.1.1 At the protein level,ELISA showed that LMP2 protein expression significantly up-regulated in ITP patients compared to healthy controls.1.2 At the mRNA level,LMP2 gene transcription up-regulated in ITP patients compared to healthy controls.However,no differences in LMP7 expression were found between two groups and LMP10 gene transcription down-regulated in ITP patients.2.ONX-0914 reduces the expression of FC yR on monocytes in ITP patients.2.1 In the monocytes of ITP patients treated with ONX-0914,FcyRIII(CD 16)expression was found to be significantly decreased,which did not happened in ITP patients treated with ML604440.2.2 ML604440 and ONX-0914 had no impact on FcyRI(CD64)expression.3.ONX-0914 inhibited the phagocytosis of macrophages to platelets:the phagocytosis of macrophages treated with ONX-0914 was lower than that of the control group,and ML604440 had no significant effect on the phagocytosis of macrophages.4.ONX-0914 Inhibited T Cell Activation:Compared with the control group and ML604440 treatment group,ONX-0914 could reduce the expression of early and late activation indexes,CD69 and CD25,of T cells.5.ONX-0914 Suppressed Thl Cell Differentiation。5.1 Compared with the control group and ML604440 treatment group,ONX-0914 could significantly reduce the differentiation of CD4+T cells from ITP patients to Th1.5.2 No significant inhibition of Th17 differentiation was observed after treatment with ONX-0914.6.ONX-0914 inhibits Thl subsets differentiation by down regulating p-STAT1 expression.Conclusion1.Compared with the control group,ITP patients had abnormal expression of immunoproteasome subunits;2.Inhibition of LMP2 and LMP7 by ONX-0914 can enhance immune tolerance by reducing the expression of activated FCyR,inhibiting the activation of T cell and the differentiation of Thl subsets.Part Ⅱ Therapeutic effect and mechanism of immunoproteasome inhibitor on ITP passive modelBackgroundIn the first part of the study,we found that the immunoproteasome subunit in ITP patients have abnormal expression.Vitro cell experiments indicate that ONX-0914 can reduce the expression of activated FCyR,inhibit T activation,inhibit the differentiation of Thl,and promote immune tolerance.To determine whether immunosuppressive agents have therapeutic effect on ITP animal models,the expression of FcγR on monocytes of spleen and liver,expression of M2 macrophages in spleen and the activation of T cells of ITP mouse were detected to further discover the specific mechanism of ONX-0914 on ITP trearment.Methods1.To establish passive ITP mice model,the mice were randomly divided into control group,ML604440 treatment group and ONX-0914 treatment group,ITP passive model mice were constructed by injecting anti-CD41 antibody into C57/BL6 mice.The control group was treated with PBS intraperitoneal injection,and the treatment group was dividedly given ML604440(10mg/kg)and ONX-0914(10mg/kg)intraperitoneal injection.The peripheral blood routine of mice was detected every other day.2.The spleen cells and liver cells were isolated and the expression of FcyR on the surface of monocytes was detected by flow cytometry;3.Immunofluorescence technique was used to detect the expression of M2 macrophages(F4/80+CD163+)in spleen of mice.4.PBMCs were isolated from peripheral blood collected from ophthalmic artery,and t he activation of CD4+ T cells isolated from PBMCs and thymocytes were detected by flow cytometryResults1.Compared with the control group and ML604440 group,OINX-0914 significantly increased the peripheral blood platelet level of ITP passive model mice.2.ONX-0914 decreased the expression of CD64 in spleen monocytes.3.ONX-0914 increased the expression of M2 macrophages in spleen.4.ONX-0914 decreased the activation of CD4+T cells.ConclusionONX-0914 has significant therapeutic effect on ITP mice,which can decrease the expression of CD64,up-regulate the proportion of M2 macrophages in spleen and inhibit the activation of CD4+T cells.These results suggest that the inhibition of immunoproteasome is a potential therapeutic approach for ITP patients. |
