Intervention Measures And Mechanism Of Macrophage Subpopulation Imbalance In Primary Immune Thrombocytopenia

Objective:Immune thrombocytopenia（ITP）is a common autoimmune disorder in children.It is primarily characterized by a reduction in platelet count,which is typically caused by an increase in immune-mediated platelet destruction and/or a decrease in platelet production.Macrophages,as an integral part of the immune system,play a pivotal role in the pathogenesis of ITP.Macrophages act both as effector cells,phagocytizing platelets,and as antigen-presenting cells,stimulating B cells to produce autoantibodies against platelets.Studies have confirmed that an imbalance in macrophage subsets is closely related to the onset and progression of ITP.In ITP,there is a phenomenon of abnormal polarization towards pro-inflammatory classically activated macrophages（M1）,and the immune response of monocyte-derived macrophages（MDMs）in patients is significantly enhanced.Therefore,in-depth exploration of the imbalance mechanism of macrophage subsets in ITP,screening out the intervention targets with potential therapeutic value,and based on this,developing effective interventions to restore the balance of macrophages and improve the clinical symptoms and quality of life of ITP patients are the hotspots and difficulties of current research.This study focused on the polarization and immune function of macrophages.NLRP3inflammasome,TNFR2,FcγRI,FcγRⅡa and FcγRⅡb of macrophages were used as the intervention targets.MCC950,Hydatid antigen B and DMF were used as intervention methods to investigate the activation level of NLRP3 inflammasome in ITP and the effect of inhibiting NLRP3-mediated inflammasome activation on M1 macrophage polarization and immune function.To investigate whether hydatid antigen B could promote M2polarization of macrophages through TNFR2 to improve ITP.Furthermore,to investigate whether hydatid antigen B could up-regulate the expression of FcγRⅡb and down-regulate the expression of FcγRI and FcγRⅡa in bone marrow macrophages,and mediate macrophage immune tolerance to alleviate ITP as an immunosuppressant.In addition,the effect of dimethyl fumarate（DMF）on ITP by regulating macrophage polarization was also investigated.Methods:Part Ⅰ:The expression of NLRP3 mRNA in peripheral blood mononuclear cells（PBMC）and CD14⁺monocytes of ITP patients（ITP group）and Control group（Control group）was detected by RT-qPCR.The levels of IL-1βand IL-18 in serum of the two groups were determined by ELISA.M0 macrophages（MDMs）from ITP group were divided into 4 groups:IgG control group（IgG group）,MCC950 treatment group（MCC950group）,LPS,IFN-γand IgG treatment group（LPS+IFN-γ+IgG group）and LPS,IFN-γand MCC950 treatment group（LPS+IFN-γ+MCC950group）;mRNA and protein levels of M1 macrophage markers CD86,iNOS and MCP-1 were detected by RT-qPCR and Western blot.Western blot was used to detect the expression of NLRP3inflammasome associated protein,ASC,Cleaved caspase-1 and IL-β.Flow cytometry was used to detect the phagocytosis of MDMs on platelets in each group,and CFSE was used to detect the proliferation of CD4⁺T and CD8⁺T.Part Ⅱ:The C57/B6 mice with wild-type（WT）and tumor necrosis factor receptor 2（TNFR2）gene knockout(TNFR2^-/-)were divided into 6 groups:WT group,WT modeling group（ITP modeling group）,WT treatment group（ITP modeling group was treated with hydatid antigen B）,TNFR2^-/-group,TNFR2^-/-modeling group and TNFR2^-/-treatment group,with 10 mice in each group.The mice were treated 1 week after modeling,and were sacrificed 2 weeks later.Body weight change,organ index,peripheral blood platelet number and blood routine indexes of each group were compared.The proportion of M2-type macrophages in peripheral blood was detected by flow cytometry.The contents of iNOS,IL-6,Arg1 and IL-10 in serum were detected by ELISA.Bone marrow derived macrophages（BMDM）were isolated and induced,and the expression levels of Arg1,IL-10,iNOS and IL-6 were detected by qRT-PCR.The BMDM of WT group and TNFR2^-/-group were respectively induced into M2-type macrophages,during which hydatid antigen B was added and divided into WT M2 group,WT M2+antigen B group,TNFR2^-/-M2 group and TNFR2^-/-M2+antigen B group.The expression levels of Arg1and IL-10 were detected by qRT-PCR.Part Ⅲ:ITP mouse model was constructed and ITP mice were treated with hydatid antigen B.E.coli prokaryotic expression system expressed HA-B in vitro.HA-B expression and protein purity were determined by SDS-PAGE gel electrophoresis and Western blot.Animal experiments were divided into control group,model group and HA-B group,n=8.Flow cytometry（FCM）was used to measure the number of peripheral blood platelet in mice.Mouse peripheral blood anti-platelet antibody IgG titer was determined by ELISA.Mouse peritoneal primary macrophages were isolated and platelet phagocytosis was performed in vitro and in vivo.HE staining and histological analysis of mouse femoral bone marrow cavity were performed.CD11b^highmacrophages in mouse bone marrow were sorted by FCM,and then CD64⁺,CD32⁺and CD16⁺cells were sorted and counted.The expression levels of FcγRI,FcγRⅡa and FcγRⅡb mRNA in bone marrow CD11b^highmonocyte/macrophages were determined by q PCR.Part Ⅳ:The Balb/c mice were divided into control group,ITP group,DMF low-dose group and DMF high-dose group,each with 15 mice.Except for the control group,the other three groups of mice were all constructed with ITP models.Mice in low-dose DMF group and high-dose DMF group were given 10 mg/kg and 20 mg/kg DMF by gavage,once a day for 14 consecutive days.The automatic blood cell analyzer was used to detect the levels of peripheral blood platelets（PLT）,red blood cells（RBC）,white blood cells（WBC）and hemoglobin（Hb）in mice;ELISA method was used to detect the levels of serum IFN-γ,IL-2,IL-4,IL-10,IL-17 and TGF-β1 in mice.Electronic balance was used to weight mouse body weight,spleen and thymus,and organ index was calculated;HE staining was used to observe the number of megakaryocytes in mouse spleen tissue and bone marrow tissues;Immunofluorescence staining was used to detect the polarization of macrophages in mouse spleen tissues;Flow cytometry was used to determine the polarization of macrophages in the peripheral blood of mice.Results:Part Ⅰ:Compared with the control group,the expression of NLRP3 mRNA in PBMC and CD14⁺monocytes,and the concentration of IL-1βand IL-18 in serum of ITP group were increased significantly.Platelet counts were negative correlated with NLRP3 mRNA expression in CD14⁺monocyte and the concentration of IL-1β,IL-18 in serum in patients with ITP,Compared with IgG group,the mRNA and protein expressions of M1macrophage markers CD86,iNOS,MCP-1,and the pr-otein expression level of NLRP3,ASC,cleaved caspase-1 and IL-β,the platelet phagocytosis and the proliferation promoting ability of CD4⁺T and CD8⁺T cells were significantly increased in LPS+IFN-γ+IgG group and LPS+IFN-γ+MCC950group.Compared with LPS+IFN-γ+IgG group,the above indexes were significantly decreased in LPS+IFN-γ+MCC950group.Part Ⅱ:.Compared with WT control group and TNFR2^-/-control group,the body weights of the mice in WT-ITP model group and TNFR2^-/-ITP model group were decreased,the spleen and thymus indexes were increased,the platelet counts and red blood cell counts were decreased,the hemoglobin levels were decreased,the white blood cell counts were increased,and the coagulation time were increased;the percentages of M2 macrophages in the peripheral blood were decreased,the levels of Arg1 and IL-10 in serum were decreased,and the levels of iNOS and IL-6 were increased;the expression levels of Arg1 and IL-10 mRNA in the BMDM were decreased,while the expression levels of iNOS and IL-6 mRNA were increased.Compared with WT-ITP model group,the body weight of the mice in WT-HA-B group was increased,the spleen and thymus indexes were decrease,the platelet count and red blood cell count were increased,the hemoglobin level was increased,the white blood cell count was decrease,and the coagulation time was decreased;the percentage of M2 macrophages in the peripheral blood was increased,the levels of Arg1 and IL-10 in the serum were increased,and the levels of iNOS and IL-6 in serum were decreased;the expression levels of Arg1 and IL-10mRNA in the BMDM were increased,while the expression levels of iNOS and IL-6mRNA were decreased.Compared with WT-HA-B group,the body weight of the mice in TNFR2^-/-HA-B group was decreased,the spleen and thymus indexes were increased,the platelet count and red blood cell count were decreased,the hemoglobin level was decreased,the white blood cell count was increased,and the coagulation time was increased;the percentage of M2 macrophages in the peripheral blood was decreased,the levels of Arg1 and IL-10 in the serum were decreased,and the levels of iNOS and IL-6were increased;the expression levels of Arg1 and IL-10 mRNA in the BMDM were decreased,while the expression levels of iNOS and IL-6 mRNA were increased.Compared with WT M2 group,the expression levels of Arg1 and IL-10 mRNA in the macrophages in WT M2+HA-B group were increased;compared with WT M2+HA-B group,the expression levels of Arg1 and IL-10 mRNA in the M2 macrophages in TNFR2^-/-M2+HA-B group were decreased.Part Ⅲ:SDS-PAGE gel electrophoresis and Western blot results showed that HA-B expression level was high with high quality and purity.On the 21st day of ITP modeling,compared with control group,platelets in model group was significantly decreased.Serum anti-platelet antibody IgG titer was significantly increased.The mean fluorescence intensity（MFI）of macrophages phagocyted platelets increased significantly in vitro and in vivo.Megakaryocytes with multi-nucleus were almost absent in bone marrow.The MFI of CD64⁺and CD32⁺in bone marrow macrophages was significantly increased,and CD16⁺was significantly decreased.The relative mRNA expression levels of FcγRI and FcγRⅡa were significantly increased and FcγRⅡb mRNA were significantly decreased.Instead,the above phenomenon were reversed after HA-B treatment,and compared with model group,all the above indexes were significantly improved in HA-B group.Part Ⅳ:After the modeling started,the number of peripheral blood platelets in the mice was significantly lower than that of the control mice,indicating that the modeling was successful;After modeling,compared with the control group,the PLT,RBC and Hb of the ITP group were decreased,WBC was increased,serum IFN-γ,IL-2 and IL-17 were increased,and TGF-β1,IL-4 and IL-10 were all de-creased,the spleen coefficient was increased,the number of megakaryocytes in the spleen and bone marrow were increased,the positive expression rate of M1 macrophage marker CD68⁺CD86⁺in spleen tissue was increased,and the positive expression rate of M2 macrophage marker CD68⁺CD206⁺was decreased,the proportion of F4/80⁺CD86⁺labeled cells in peripheral blood was increased,and the proportion of F4/80⁺CD206⁺labeled cells was decreased;Compared with the ITP group,the PLT of the mice in the DMF low-dose group was increased,and the WBC was decreased,the PLT,RBC and Hb of the DMF high-dose group mice were increa-sed,while the WBC was decreased,in the DMF low-and high-dose groups,the serum levels of IFN-γ,IL-2 and IL-17 were all decreased,TGF-β1,IL-4 and IL-10were all increased,spleen coefficient was decreased,the number of megakaryocytes in the spleen and bone marrow was also decreased.In addition,the positive expression rate of CD68⁺CD86⁺in the spleen tissue was decreased,and the positive ex-pression rate of CD68⁺CD206⁺was increased,the proportion of F4/80⁺CD86⁺la-beled cells in the peripheral blood was also decreased.the proportion of F4/80⁺CD206⁺labeled cells was increased.Conclusion:Part Ⅰ:The activation level of NLRP3 inflammasome in ITP is abnormally elevated,which was related to the excessive M1 polarization of MDMs.Inhibiting NLRP3 mediated inflammasome activation could attenuates the M1 polarization and immune function of macrophages.Part Ⅱ:Hydatid antigen B can promote the macrophage M2 polarization through TNFR2,thereby exerting the therapeutic effect on ITP.Part Ⅲ:Hydatid antigen B may up-regulate the expression of FcγRⅡb and down-regulate the expression of FcγRI and FcγRⅡa in bone marrow macrophages,so as to regulate the recognition and phagocytic activity of macrophages to platelet antigens,and mediate macrophage immune tolerance as an immunosuppressant to alleviate immune thrombocytopenia.Part Ⅳ:DMF can increase the number of PLT in ITP mice,reduce the increase of megakaryocytes in the spleen and bone marrow,and regulate the level of cytokines,thereby improving ITP.Its mechanism may be related to inhibiting the differentiation of macrophages into M1 type and promoting the differentiation to M2 type.