Novel Murine Model Of Immune Thrombocytopenia Through Immunized CD41 Knockout Mice And The Effect Of Human Leukocyte Antigen-G In Immune Thrombocytopenia

Part I Novel Murine Model of Immune Thrombocytopenia th rough Immunized CD41 Knockout MiceImmune thrombocytopenia(ITP)is a common autoimmune hemorrhagic disease characterized by decreased platelet counts and an increased bleeding risk.Glucocorticoid and immunoglobulin are the first-line treatment for the new-diagnosed patients.Although platelet-promoting agents,monoclonal antibody(anti-CD20),immunosuppressors and splenectomy bring hopes to the patients,in about 1/3 of the patients,symptoms can not be controlled by these therapies,and progress at last.To date,many murine models of ITP have been used to elucidate the pathogenesis of this disorder and explore the novel therapeutics.These models can be divided into three basic categories:passive ITP,secondary ITP and platelet-induced ITP.The first two types are not ideal for chronic ITP pathophysiology in which both T cell and B cell autoimmune attacks are focused on platelet-specific antigens.In view of this,Semple developed a brilliant murine model of severe chronic ITP with the use of immunized CD61-KO mice.This model explicitly demonstrated the immunopathogenesis of chronic ITP,mimicked the human disease and correlated quite well with clinical ITP.However,placental defects are evident in 25%of CD61-KO females and post-natal hemorrhage does occur,reducing the survival rate of embryos and pups and increasing the cost.We want to develop another platelet-induced ITP model through CD41 KO mice,since CD41 is a classical marker to megakaryocytes/platelets.Objective:To explore the feasibility of developing an ITP model through CD41 KO mice.Methods:1.Placenta and fetal mice were detected at E14,neonatal mice were detected at D3.2.CD41 KO mice were immunized with platelets of wide-type(WT)mice,and then their splenocytes were injected into SCID mice.The platelet counts,the antibody levels and the megakaryocyte counts in the bone marrows were detected.3.CD41 KO mice were immunized with platelets of WT mice,after lymphocyte-depletion,splenocytes with different cellular constituent were injected into SCID mice.The platelet counts,the antibody levels and the megakaryocyte counts in the bone marrows were detected.4.CD41 KO mice were immunized with platelets of WT mice,and then their splenocytes were injected into splenectomized SCID mice.The platelet counts,the antibody levels were detected.Results:1.Compared with WT mice,the placenta of CD41 KO mice were not different at E14.There was no hemorrhage in the fetal mice and the number of the fetal mice did not decrease.There was also no hemorrhage in the neonatal mice at D3.2.Compared with control SCID mice,there was obvious decrease of the platelet counts,obvious increase of the antibody levels and obvious decrease of the megakaryocyte counts in the morbid SCID mice.3.The mature megakaryocyte counts of SCID mice increased by transferred with the CD8+ cell-depleted splenocytes,indicating that CD8+ T cells played important roles in decrease of mature megakaryocytes of ITP.4.After splenocytes transfer,the antibody level was obviously decreased in the splenectomized SCID mice.Their platelet counts also increased,but could not restore.Conclusions:1.Platelet-induced ITP models can better explore the mechanism of chronic ITP.2.CD41 KO mice can also be used to develop another efficient platelet-induced ITP model.Part ? the Effect of Human Leukocyte Antigen-G in ImmuneThrombocytopeniaImmune thrombocytopenia(ITP)is a common auto-immune bleeding disease characterized with increased platelet destruction and decreased thrombocytopoiesis.Although the etiology of ITP is still unclear,the loss of immune tolerance to platelet surface antigen is the key question.The number and function of various immunoregulatory cells are abnormal,including T cells,B cells,dendric cells(DCs),myeloid-derived suppressor cells(MDSCs),resulting in increased platelet destruction and decreased thrombocytopoiesis.Human leukocyte antigen-G(HLA-G)is a non-classical major histocompatibility complex class I antigen.Compared with classical HLA-I,the polymorphism of HLA-G is low,with 4 membrane-bound forms(HLA-G1,HLA-G2,HLA-G3,HLA-G4)and 3 soluble forms(HLA-G5,HLA-G6,HLA-G7).HLA-G exerts its immunoregulatory functions through combining with the inhibitory receptors,including immunoglobulin-like transcript 2(ILT2/LILRB1/CD85j),immunoglobulin-like transcript 4(ILT4/LILRB2/CD85d)and killer cell immunoglobulin-like receptor(KIR2DL4/CD158d).ILT2 was expressed on T cells,B cells,some natural killer cells(NKs)and DCs.ILT4 was expressed on myeloid cells,including monocytes and DCs.KIR2DL4 was mainly expressed by NKs of the uterus.Lower soluble HLA-G(sHLA-G)level was found in patients with rheumatoid arthritis and systemic lupus erythematosus,and was associated with disease activity in both.Moreover.less or dysfunctional ILT2 and ILT4 were involved in the pathogenesis of systemic lupus erythematosus.Another study by Kim et al showed that functional HLA-G 14-bp polymorphism was associated with susceptibility to rheumatoid arthritis and systemic lupus erythematosus.However,the role of HLA-G in ITP remains obscure.Objective:To explore the relationship of HLA-G and ITP.Methods:1.Specimen collection:30 ITP patients and 15 healthy controls were enrolled and their peripheral blood was collected.2.Plasma and lymphocytes/monocytes preparation:plasma and peripheral blood monocytes(PBMCs)were collected by density gradient centrifugation;CD4+ T cells and CD 14+ monocytes were collected by magnetic activated cell sorting(MACS).3.The level of anti-platelet antibodies was determined by modified monoclonal antibody-specific immobilization of platelet antigen(MAIPA).4.The level of HLA-G in serum was determined by enzyme-linked immunosorbent assay(ELISA).5.The level of multiple cytokines was determined by Bio-Plex.6.Cell culture:CD14+ monocytes were cultured with granulocyte-macrophage colony stimulating factor(GM-CSF)and IL-6 for 5 days,then added with lipopolysaccharide(LPS)/or recombined human HLA-G(rhHLA-G)for another 2 days.CD4+ T cells were cultured with anti-CD3 antibodies and recombinant human IL-2(rhIL-2).then added with some DCs/or rhHLA-G for 5 days.Results:1.Compared with the healthy controls,the level of HLA-G of the ITP patients positive for anti-platelet antibodies was significantly decreased,while no obvious difference was found in the ITP patients negative for anti-platelet antibodies.2.Compared with the healthy controls,the level of mHLA-G on the CD4+ T cells and CD14+ monocytes of ITP patients were decreased,the same as the ILT2 on the CD4+T cells and ILT4 on the CD 14+ cells.The rhHLA-G protein could upregulate the above ILTs.3.rhHLA-G could reprogram the cytokines secreted by PBMCs:after adding with the rhHLA-G protein,the level of TNF-?,IL-12 and IL-17 was significantly decreased,while the level of IL-?,IL-2,IL-4,IL-10,G-CSF and GM-CSF was significantly increased.4.Dendritic cells induced from ITP patients express higher levels of CD80 and CD86 than those from healthy controls.In the presence of rhHLA-G,CD80 and CD86 expression was significantly downregulated both in ITP patients and healthy controls.In vitro-generated dendritic cells from ITP patients stimulated significantly more CD4+ T cell proliferation compared with those from healthy controls.However,rhHLA-G-modulated dendritic cells from ITP patients suppressed CD4+ T cell proliferation by 38.8%compared to unmodulated cellsConclusions:1.The level of HLA-G and ILTs of ITP patients was decreased,while rhHLA-G could increase the expression of ILTs.2.rhHLA-G could reprogram the secreted cytokines,suppress the maturation of DCs and proliferation of T cells.3.HLA-G may play an important role in the pathogenesis and treatment of ITP.