| Introduction:Dendritic cells (DCs) are versatile regulators of the immune homeostasis, primarily influencing T cells. The DCs that induce autoimmune tolerance are called tolerogenic DCs (tDCs). The tDCs express low levels of MHC II molecule and costimulatory molecules and exhibit potent immunoregulatory abilities in autoimmune diseases.Immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disease characterized by immune-mediated platelets destruction and/or suppression of platelets production, in which autoreactive T cells that recognize and respond to autologous platelet antigens are activated resulting in the breakdown of the immune tolerance to platelet autoantigens. As a promising immunotherapeutic strategy for autoimmune disorders, tDCs have received considerable attention. tDCs has been already sueeessfully tested in a number of experimental models of autoimmune diseases including experimental autoimmune encephalomyelitis (EAE), collagen and adjuvant-induced arthritis(CIA), diabetes in the non-obese diabetie (NOD) mouse.Our study generated tDCs stimulated by several cytokines in vitro and develop an animal model that simulate human ITP based on the foreign experience and to evaluate the effect of tDCs on this experimental model.Objective:1. To establish the culture method of tolerogenic dendritic cells(tDCs)in vitro, followed by identification of its morphology、immunological phenotype and metergasis, Looking for the appropriate cytokines needed for cell culture;2. To develop an ideal animal model that simulate human ITP, followed by identification of its clinical indicators,for example the average number of platelets, platelet volume;3. To investigate the effect and its mechanism of TGF-beta 1 and IL-10 induced tDCs in ITP mice model.Methods:1. The culture of tDCs:C57BL/6 mice are used. Tolerogenic dendritic cells were generated from bone marrow precursors cultured with GM-CSF、IL-4、TGF-β1 and/or IL-10. The cells were devided into five groups:imDC group(GM-CSF+IL-4)、mDC group (GM-CSF+IL-4+LPS)、10-DCs group (GM-CSF+IL-4+IL-10)、T-DCs group (GM-CSF+IL-4+TGF-β1)、lOT-DCs group (GM-CSF+IL-4+IL-10+TGF-β1).tDCs were marked by GFP lentivirus.The cell morphological differences were examined under optical microscope and Fluorescence microscope. The differences on cell phenotype were analyzed by flow cytometry(FCM). The capability to stimulate the proliferation of lymphocytes were examined through WST method. The mRNA expression of IL-12 and CCR7 were measured by qRT-PCR method. IL-10 and TGF-P of the cell culture medium and IFN-γ、IL-10 and TGF-βof the culture medium in mixed lymphocyte culture were measured with enzyme linked immunosorbent assay(ELISA).2. TheITP animal model:BALB/c mice were immunized once intraperitoneal injection of MWReg30 antibody or PBS.The Platelet count and Mean platelet volume(MPV)were measured by automated cytometer at 1h,3h,24h,48h,72h, 96h,120h after immunized. Presence of Platelet-associated immunoglobulin G (PAIgG)was analyzed by flowcytometry(FCM).We also detected the plasma levels of IFN-γ、IL-2、IL-4、IL-10 and TGF-βby ELISA. In addition,bone marrow examination was performed,in order to observe the morphocytology of megakaryocytes.3. The role of tDCs in the theropy of ITP animal model and its mechanism:The ITP animal transfusion experiment were divided into two groups:theropy group(transfusion tDCs)、control group(transfusion PBS).the cell or PBS therapy were performed at 1h after injection of antibody. Using GFP to mark tDCs for site-specific study. The Platelet count was measured at 1h,3h,24h,48h,72h,96h, 120h after immunized. We also detected the plasma levels of IFN-γ、IL-2、IL-4、 IL-10 and TGF-βby ELISA. Proportion of CD4+CD25+Foxp3+ Treg cells in peripheral blood was analyzed by FCM.Result:1. Tolerogenic dendritic cells were generated from bone marrow precursors cultured with GM-CSF、IL-4、IL-10 and/or TGFp.2. The characteristic of several tDCs:1) Compared to the mDCs, The 10-DCs highly express the specific marker CD11c and inhibitory molecular PD-L1 and ILT3, lowly express CD80、CD86 and I-A/I-E, induce light lymphocytes proliferation, the decrease secretion of IL-12 and CCR7, and the increase secretion of IL-10 and TGFβin the culture medium, in the coculture medium with mixed lymphocytes, IFN-yis decreased, IL-10 is increased, the level of TGFβ does not change significantly.2) Compared to the mDCs, The T-DCs highly express the specific marker CDllc and inhibitory molecular PD-L1 and ILT3, lowly express CD80 and I-A/I-E, induce normal lymphocytes proliferation, the normal secretion of IL-12 and CCR7, and the increase secretion of TGFβ in the culture medium, in the coculture medium with mixed lymphocytes, IFN-yis decreased, IL-10 and TGFβ is increased.3) Compared to the mDCs, The 10T-DCs highly express the specific marker CD11c and inhibitory molecular PD-L1 and ILT3, lowly express CD80、 CD86 and I-A/I-E, induce light lymphocytes proliferation, the decrease secretion of IL-12 and CCR7, and the increase secretion of IL-10 and TGFj3in the culture medium, in the coculture medium with mixed lymphocytes, IFN-yis decreased, IL-10 and TGFβ is increased.3. Making ITP models and detection:1) tDCs present green after transfection of GFP by lentivirus without the change of cell phenotype.2) After the MWReg30 administration, Platelet count of the mice showed a significant decrease at 1h (P<0.05), and went back to the normal at 120h.3) When making model, significant increase in MPV was observed with decreasing platelet count at 24h (P<0.05), reached a maximum at 48h and continued to 120h (P<0.001). There is a negative correlation relationship between MPV and Platelet count. Bone marrow examination revealed normal numbers of megakaryocytes with no dysplastic features on light microscopy.4) Significant increase in PAIgG was observed compared to control group.5) The Plasma levels of IFN-y and IL-4 were found to be significantly increased in ITP mice, compared to control animals. IFN-yshowed significantly change at 3h after injection of MWReg30 (P<0.05),and went back to the normal at 120h. IL-4 showed significantly change at 24h after injection of MWReg30 (P<0.05)and went back to the normal at 96h. The Plasma levels of IL-2、 IL-10 and TGF-β had no significantly changes.4. The role of tDCs in the theropy of ITP animal model and its mechanism:1) Platelet count of the tDCs-therapy mice showed a significant increase compared to control group.2) The Plasma levels of IFN-yin tDCs-therapy mice were found to be significantly decreased compared to control group at 3h、24h、48h、72h after injection of MWReg30 (P<0.05). The Plasma levels of IL-4、IL-2、IL-10 and TGF-β had no significantly changes.3) The green cells were found in spleen of tDCs-therapy mice by Fluorescence microscope. 4) Proportion of CD4+CD25+Foxp3+ Treg cells in peripheral blood of tDCs-therapy mice was significantly increased compared to control group.Conclusions:1. The best appropriate tolerogenic dendritic cells were generated cultured with GM-CSF、IL-4、IL-10 and TGFβ.2. A murine model bearing much resemblance to the course and Pathophysiology of human ITP could serve as a Potential tool for the research of ITP.3. Transfused tDCs migrated to the spleen and played the therapy role via tail vain. Platelet count showed a significant increase, the plasma levels of IFN-γwere found to be significantly decreased in tDCs-therapy mice compared to control group.4. The mechanism of the treatment could be the increased CD4+CD25+Foxp3+Treg cells intDCs-therapeutic ITP mice. |
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