The Effects Of Regulatory Dendritic Cells In Primary Immune Thrombocytopenia

Primary immune thrombocytopenia (ITP) is the most common clinical hemorrhagic disease, taking up nearly30%of total hemorrhagic diseases. ITP clinical manifestations include skin mucosa bleeding, menorrhagia, internal bleeding, and even intracranial hemorrhage, seriously affecting human health. Pathogenesis of this disease is mainly the immune tolerance imbalance, generating glycoprotein (GP)-specific autoantibody, mostly GPâ…¡b/â…¢a and/or GPIb/â…¨ IgG Such autoantibodies combine with GP antigens of platelet membrane, leading to the destruction of platelet by mononuclear phagocytes system. Mechanism of abnormal immune reaction includes complex interactions among antigen presenting cells, T cells and B cells, such as imbalance of Thl/Th2cell subsets, activation of platelet-specific autoreactive T cells and B cells, reduction of in the number of regulatory T cells or impaired inhibition function, and platelet destruction by cytotoxic T cells. However, the destruction of immune tolerance to platelet autoantigen and mechanism of autoantibody production are not clear.Studies show that T cells with potential autoreactive activity can be found in healthy adult individuals. These autoreactive T cells can maintain the state of autoimmune tolerance by a variety of mechanisms, including inducing apoptosis and immune incompetence. In this process, indoleamine2,3-dioxygenase (IDO) and dendritic cells (DCs) with immunoregulation function play a very important role.IDO is a monomeric protein containing ferrihemoglobin, which is the only limiting velocity enzymes (except for liver) that can catalyze epoxidation cracking of benzpyrole in tryptophan molecules so as to be catabolized along the kynurenic acid pathway. IDO specifically expresses on macrophages and DCs. IDO can affect functions of lymphocytes by degradation of tryptophan in local tissue, playing an important role of metabolic immune regulation in tumor escape, maternal-fetal tolerance, chronic infection, autoimmune diseases, and transplantation tolerance. According to recent studies, abnormalities of IDO-mediated tryptophan catabolism exist in many autoimmune diseases, such as multiple sclerosis, autoimmune diabetes mellitus, diffuse toxic goiter, and rheumatoid arthritis.DCs are known as the most important antigen-presenting cell (APC) in vivo currently and the only antigen-presenting cell that activates naive T cells, which occupy unique position in induction of immune response. Recent studies show that DCs involving in not only immune response of foreign antigens, but also immune tolerance of autoantigen. Recent studies also show that the immunomodulatory function of DCs is closely associated with functional IDO expression.Studies show that IDO plays a very important role for DCs to maintain homeostasis and immune tolerance, regulatory DCs express high level of functional IDO, and the IDO activation in the immune response of exogenous antigen can induce T cell apoptosis so as to maintain the stability of immune environment. In autoimmune diseases, DCs expressing a high level of functional IDO could induce autoreactive lymphocyte apoptosis and activation of regulatory T cells. This is an important mechanism to maintain peripheral immune tolerance. In addition to the level of IDO expression in cells, immune regulatory function of regulatory DCs is also closely associated with IDO enzyme activity. IDO expression can be induced by immune-inflammatory mediators, such as IFN-y, IFN-a, and tumor necrosis factor (TNF). The combination of B7-1/B7-2molecules and CTLA-4/CD28is an essential condition to initiate IDO enzymatic activity of DCs. When such a combination is disconnected, IDO is in the inactive state, and cannot decompose tryptophan and inhibit T cell proliferation. Another study shows that soluble CTLA-4can induce human mature DCs to express IDO with enzymatic activity, recombine human CTLA-4-of Ig Fc fusion protein, have a high affinity with co-stimulatory molecule B7-1, B7-2, and lead to the lack of tryptophan in T-cell micro-environment and accumulation of kynurenine, thus inhibiting T cell proliferation and apoptosis of lymphocytes. In addition, CTLA-4-Ig-induced DCs expressing enzyme activity IDO can also promote the generation of regulatory T-cells (Tregs) thereby inhibiting effector T cell activity and inducing immune tolerance. In our previous study, it can also be found that CTLA-4-Ig can induce autoreactive T cells of ITP patients to produce specific immune tolerance by a cell-dependent manner in vitro. The above research findings offer an important basis for the application of the CTLA-4-Ig induced DCs to treat autoimmune thrombocytopenic purpura.Objective:To detect IDO expression in ITP patients.CTLA-4-Ig induces peripheral blood DCs of ITP patients to express IDO with enzyme activity in vitro, and we detect IDO expression and activity changes.To study immune tolerance of IDO+DC by examining IDO+DCs'effects on proliferation, activation, apoptosis of T cells and regulatory T (Treg) cells.Materials and Methods:Selection of cases and normal controls:15cases of ITP patients, including five newly diagnosed ITP patients and10recurrent ITP patients. Normal controls come from12healthy volunteers.Separate mononuclear cells (PBMCs) from peripheral blood of ITP patients, use GM-CSF, IL-4to culture7d. For cultured immature DCs, TNF-a,IL-6, IL-1β, PGE2, etc. are used to culture2days. Cultured mature DCs are divided into two groups:group1is added with CTLA-4-Ig (10μg/ml), and group2is added with CTLA-4-Ig (0μg/ml).Flow cytometry is used to detect IDO expression in PBMCs and mature DCs, reverse transcriptase polymerase chain reaction (RT-PCR) is used to detect expression of IDO mRNA, and Western boltting is used to detect IDO protein expression.High Performance Liquid Chromatography (HPLC) is used to detect the concentration of Trp and its specific metabolite Kyn of ITP patients and normal controls so as to determine the enzyme activity of IDO.Two groups of induced DCs form mixed lymphocyte reaction (MLR) system with platelet-specific T lymphocytes respectively. Detect T lymphocyte proliferation in the system, and flow cytometry is used to detect proliferation, activation, and apoptosis of T lymphocytes, and the proportion of Treg cells.Results:IDO expression in DCs of ITP patients Compared with normal controls, mean fluorescence intensity (MFI) of IDO was significantly reduced in DCs of ITP patients (376.9±28.00vs124±23.10, P <0.01).Impacts of CTLA-4-Ig on IDO expression and activity in DCs of ITP patients Group1(CTLA-4-Ig,10μg/ml), and Group2(CTLA-4-Ig,0μg/ml). The relative expression level of IDO mRNA in group1is36.8times higher than that of Group2(p=0.0004). Western Blotting and flow cytometric analysis also proved that expression level of IDO in group1is significantly higher than that of group2(401.0±44.9vs.124.0±23.1; P<0.0001). HPLC measured the kynurenine concentration in DCs culture supernatant. The kynurenine in group1is significantly higher than that of group2(0.86±0.08uM vs.0.39±0.03uM; P=0.0179). After adding the IDO specific inhibitor1-MT, its kynurenine concentration in supernatant significantly decreased (0.86±0.08uM vs.0.19±0.05uM;P=0.0108).Immune tolerance effect of CTLA-4-Ig inducing DCs'functional IDO high expressiona) Inhibition effect of IDO+DCs on T cell proliferation:Compared with DCs not induced by CTLA-4-Ig, CTLA-4-Ig induced IDO+DCs significantly inhibited the proliferation of platelet-specific T cells (0.43±0.10vs.0.65± 0.06; P<0.0001); however, after1-MT is added to deny IDO activity, inhibition effect of CTLA-4-Ig induced IDO+DCs on platelet-specific T cell proliferation was significantly reduced (0.74±0.04vs.0.43±0.10; P <0.0001).b) Inhibition effect of IDO+DCs on T cell activation:flow cytometry is used to detect expression of T cell surface activation signs-CD25, CD69and CD71on T cells. It is found that proportions of CD3+/CD25+cells, CD3+/CD69+cells, and CD3+/CD71+cells in group1are22.7%±4.1%,8.0%±6.5%and24.4%±10.4%, and the proportions in group2are31.0%±4.7%,15.0%±1.0%,32.0%±12.5%.c) Promotion effect of IDO+DCs on lymphocyte apoptosis:flow cytometry is used to analyze effect of CTLA-4-Ig induced IDO+DCs on apoptosis of lymphocytes. Compared with group2, lymphocyte apoptosis in group1significantly increased. Annexin V%of CD4+cells, CD8+cells, and CD19+cells in group1are21.8%±7.8%,10.7%±3.1%and18.9%±9.9%, which are significantly higher than those in group2(13.8%±6.1%,7.8±2.9%, and13.3%±9.3%).d) IDO+DCs can induce generation of regulatory T cells (Treg):according to the detection of proportion of CD4+CD25+Foxp3+(Treg) cells, it can be seen that proportion of Treg cells in group1is significantly higher than group2(5.8%±1.7%vs.2.7%±0.9%; P=0.0001).Conclusions:IDO expression in DCs of ITP patients was decreased.CTLA-4-Ig enhanced IDO expression and activity in DCs of ITP patients. CTLA-4-Ig induced high expression of IDO in DCs:IDO+DCs inhibited T cell proliferation; inhibited T cell activation; promoted apoptosis of lymphocytes; induced generation of regulatory T cells (Treg).In summary, IDO expression and activity of ITP patients are abnormal, and the IDO-mediated tryptophan catabolism destruction is one of the pathogenic mechanisms of ITP. CTLA-4-Ig can increase DCs functional IDO expression of ITP patients, enhance IDO enzymatic activity, inhibit proliferation and activation of T cells, promote lymphocyte apoptosis, and increase the proportion of Treg cells. Moreover, the above actions are IDO-dependent. IDO-mediated tryptophan catabolism may play an important role in the onset of ITP, providing ITP treatment with a new approach. Primary immune thrombocytopenia (ITP) refers to thrombocytopenia caused by excessive destruction of platelets in the reticuloendothelial system due to loss of immune tolerance and the combination of produced anti-platelet autoantibody and platelet surface-specific antigen. In addition, a variety of cellular immune mechanisms are abnormal. Thl polarization, reduction in number of regulatory T cells or inhibition function defects, and cytotoxic T lymphocyte (CTL)-mediated platelet destruction play an important role in the pathogenesis of ITP. So far, reasons for the above anomalies have not been clear.At present, experimental studies of ITP are mostly about clinical trials, pharmacotherapy and so on. There are no ideal animal models, resulting in that conclusions of many in vitro tests can not be confirmed by in vivo tests. Therefore, ideal animal models need to be established. In this experiment, rat platelets were used to immune CBA mice, and antigens were inputted to induce model rats to produce immune response. The slow reduction in number of platelet in CBA mice peripheral blood can be found, indicating that the model can better simulate the sustained reduction of in vivo platelet of ITP patients, and provide a new method in the study of ITP pathogenesis.In recent years, people has paid attention to role of toll-like receptors (TLRs) in mediated innate immunity and acquired immune response. At present, a total of13kinds of TLRs family members have been found. TLR1-11express in humans, which are mainly distributed on surfaces of immune cells, including dendritic cells and macrophages. TLRs are activators of immune cells and key amboceptors of innate immune system, which can regulate the activity of the adaptive immune system. Evidences show that TLRs plays a role in immune and inflammatory diseases. TLR7of intracellular expression are involved in activation of APC and production of autoantibodies. Studies have found that the combination of TLR7and specific ligand can promote activation and proliferation of B cells and the production of pathological antibodies. Moreover, TLR7levels in ITP patients have been increased, but the transduction pathway is still under exploration.Some studies suggest that endogenous costimulatory molecules involve in regulation of B cells, and the B cell activating factor (BAFF) is a newly found cell costimulatory molecule. BAFF is mainly expressed by mononuclear cells, such as dendritic cells, macrophages, and monocytes. BAFF has three kinds of receptors: transmembrane activation and calcium-modulatorand cyclophilin ligand interactor (TACI), B cell maturation antigen (BCMA), and B cell activating factor (BAFF-R). These three kinds of receptors all express on B cell surface. After combining with receptors, BAFF can activate NF-κB signaling pathway, regulate downstream gene expression, and thereby promote proliferation and activation of B cells and production of autoantibody by recruiting TRAF. Thus, BAFF plays an important role in the regulation of autoimmune diseases. However, the mechanism of BAFF in ITP patients has not been elucidated.In summary, TLRs and BAFF are involved in the process of B cell activation and antibody production, which are one of pathogenic mechanisms of autoimmune diseases. Some scholars have studied on whether there are interactions between TLRs and BAFF. In vivo experiments conducted by some scholars have proved that reactive oxygen species (ROS) can enhance mRNA and protein expression of BAFF in mouse spleen by activating TLRs signaling pathway.According to literature of ITP and TLR7, TLR7and BAFF, BAFF and B cells, and B cells and ITP, TLR7/BAFF/BAFF receptor signaling pathway may play an important role in pathogenesis of ITP. In the present study, we explored such a hypothesis:TLR7in APC affects BAFF secretion, and acts on B cells by combining with BAFF receptors, thereby affecting the production of autoantibodies. The results of this study allow TLR7to be associated with disease activity, describing the role of TLR7/B AFF/B AFF receptor signaling pathway in the pathogenesis of ITP. Objective:Establish the ITP animal model, and detect in vivo platelet count and surface-associated antibodies of model rats. Use the ITP mouse mode and control mice to detect TLR7, BAFF, and BAFF receptor expression of their splenic mononuclear cells; use TLR7imiquimod and TLR7silencing lentivirus to study effects of TLR7on mouse platelet count, PAIgG level, BAFF level, and BAFF receptor. Explore the role of TLR7/BAFF/BAFF receptor signaling pathway in the pathogenesis of ITP.Materials and Methods:Use platelet of normal Wistar rats to immunize CBA mice, and induce model rats to produce immune response by inputting antigen. Then, use blood analyzer to detect number of platelets in peripheral blood of CBA mice, and use flow cytometry to detect associated antibodies of platelet surface.Adopt lentiviral silencing vector expressing GFP for mouse TLR7(NM₁33211), and use fluorescence microscopy to observe GFP expression, assess transfection efficiency, and collect RAW264.7cells. Then, detect mRNA level of TLR7in cells by real-time PCR, and use Western blotting to detect protein level of TLR7in cells.Take mice with lowest platelet count three weeks after the first immunization as "ITP mice". Further divide ITP mice and control mice into four groups. Group1: ITP mice are injected with rat platelet (n=10), and control mice are injected with saline (n=8); group2:ITP mice are injected with rat platelet (n=8), control mice are injected with saline (n=8), and tails of mice are injected with107TU (transduction unit, TU) empty lentiviral vector; group3:ITP mice are injected with rat platelet (n=8), control mice are injected with saline (n=8), and tails of mice are injected with107TU lentiviral silencing vector; group4: ITP mice are injected with rat platelet (n=8), control mice are injected with saline (n=8), and they are injected with25μg imiquimod (TLR7agonist) every other day.Realtime fluorescence quantitative PCR is used to detect TLR7mRNA expression of mice; Western blotting is used to detect protein concentration; enzyme-linked immunoadsorbent assay (ELISA) is used to detect serum and cell supernatant BAFF level; flow cytometry is used to analyze median fluorescence intensity (MFI) in order to detect PAIgG, BAFF receptor expression.Results:Detection of platelet count and platelet surface antibody of model mice Compared with the controls, platelets in peripheral blood of model mice have gradually reduced (P<0.05) since the first week after immunization, which were minimized (P<0.01) three weeks after immunization. Then, platelet count began to slowly pick up. The further study was conducted by taking model mice with lowest platelet count three weeks after immunization as ITP mice.Expression of in vivo TLR7, BAFF, and BAFF receptors of micea TLR7expression in mononuclear cells of mouse spleen:compared with controls, TLR7mRNA expression in ITP mice increased by3.14times(P=0.003); TLR7protein level of ITP mice also significantly increased (p=0.033).b BAFF expression in mouse spleen mononuclear cells and serum:Compared with controls, the BAFF level in SMCs supernatant of ITP mice increased significantly (ITP mice:766.09±43.03pg/ml, controls:519.19±43.95pg/ml, P=0.004).Serum BAFF levels in test mice and controls before immunization are7015.27±454.33pg/ml and7043.45±435.89pg/ml. There are no significant differences between two groups (P=1). Serum BAFF level of ITP mice three weeks after the first immunization is significantly higher than that before immunization (12448.34±695.23pg/ml vs.7015.27±454.33pg/ml., P=0.000). There are also significant differences (P=0.000)) between ITP mice (12448.34±695.23pg/ml) and controls(7920.0±375.15pg/ml). There are no significant differences in controls (P=0.224) before saline injection and three weeks after saline injection. Four weeks after the first immunization, serum BAFF level of ITP mice increased significantly compared to the controls (ITP mice:8481.66±211.54pg/ml, control mice:7463.67±198.55pg/ml, P=0.006).c BAFF receptor expression in mononuclear cells of mice spleen:compared with controls, among three receptors in ITP mice, only BAFF-R was significantly increased (P=0.006), and no significant changes in levels of BCMA and TACI are found.Detection of transfection efficiency of silencing lentivirusAccording to the observation by fluorescence microscopy, more than90%of the RAW264.7cells were transfected three days after transfection, and RAW264.7cells were collected. Compared with controls, the TLR7mRNA level in cells was decreased significantly, which is only3.82±0.62%(p=0.000); according to the protein detection of TLR7in cells, TLR7silencing lentivirus can effectively interfere with TLR7protein expression in RAW264.7cells (p=0.048).Effect of TLR7on platelet countTLR7silencing lentivirus significantly increased the relative platelet count (1.11±0.12vs.0.84±0.09, P=0.025), while imiquimod significantly reduced relative platelet count in ITP mice (0.59±0.05vs.0.84±0.09, P=0.034). However, there are no significant changes in controls.Effect of TLR7on PAIgG levelFour weeks after first immunization, the level of PAIgG was significantly increased in ITP mice compared wit controls (27.44±3.68vs.3.00±0.43, P=0.002). Imiquimod significantly increased PAIgG level (56.63±13.20, P <0.001), while TLR7silencing lentivirus significantly reduced PAIgG level in ITP mice (9.99±2.78, P=0.026). No significant differences in PAIgG level are found in four controls.Effect of TLR7on BAFF levelImiquimod significantly increased BAFF level in SMCs supernatant of ITP mice (1145.17±123.70pg/ml vs.766.09±43.03pg/ml., P<0.001), but such changes are not observed in controls; TLR7silencing lentivirus did not significantly change BAFF level in ITP mice and controls. Imiquimod significantly improved BAFF level of serum BAFF in ITP mice (10578.43±417.54pg/ml, P=0.001), while TLR7silencing lentivirus reduced BAFF level in serum of ITP mice (7258.36±359.17pg/ml, P=0.011); there are no significant differences in results of four controls.Effect of TLR7on BAFF receptor Flow cytometry results show that there are no significant differences in BAFF-R level after stimulating or inhibiting TLR7in ITP mice or control mice.Conclusions:The ITP mouse model for the experiment can not only simulate the thrombocytopenia in pathogenesis of ITP, but also significantly increase PAIgG level in model mice.TLR7level in ITP mice is increased; BAFF level is increased; among three receptors of BAFF, only BAFF-R expression is increased.TLR7activation in ITP mice may negatively regulate platelet count of ITP mice, which is associated with disease activity.TLR7in ITP mice can promote PAIgGTLR7in ITP mice can enhance the secretion of BAFF.There is no direct interaction between TLR7and BAFF-R of IPT mice.In summary, the ITP mouse model established for the experiment can simulate the pathological process of thrombocytopenia in pathogenesis of ITP, providing in-depth study of ITP pathogenesis with a good animal model. TLR7/BAFF/BAFF-R signal transduction pathway may play a role in pathogenesis of ITP. Blocking TLR7/BAFF/BAFF-R signal transduction pathway can provide the treatment of ITP with a new method. BackgroundIndoleamine2,3-dioxygenase (IDO) expression in dendritic cells (DCs) can induce or maintain peripheral immune tolerance. Impaired IDO-mediated tryptophan catabolism has been observed in autoimmune diseases.Materials and MethodsThe concentrations of kynurenine were detected by high-pressure liquid chromatography. The expressions of IDO were analyzed by flow cytometry and western blot analysis. The effects of IDO+DCs stimulated with CTLA-4-Ig on T-cells proliferation and activation, lymphocyte apoptosis and Tregs were measured by flow cytometry.ResultsThe expression of IDO in DCs of ITP patients was significantly decreased. CTLA-4-Ig significantly increased the expression of functional IDO in DCs of ITP patients. IDO+DCs stimulated with CTLA-4-Ig suppressed T-cells proliferation and activation, promoted lymphocyte apoptosis and increased the percentage of Tregs.ConclusionDecreased IDO expression in DCs may play a critical role in ITP. CTLA-4-Ig successfully corrected the disorder of IDO expression in ITP. IDO+DCs stimulated with CTLA-4-Ig inhibited immune responses by an IDO-dependent mechanism. Increasing the expression and activity of IDO in DCs might be a promising therapeutic approach for ITP. Immune thrombocytopenia (ITP) is an autoimmune disorder characterized by anti-platelet autoantibody-mediated platelet destruction. Antigen-presenting cell (APC) dysfunction is considered to play crucial roles in ITP. However, how APC affects autoreactive B cells in ITP is still unknown. Using a mouse model of immune thrombocytopenia, we demonstrated an increase in levels of TLR7in splenic mononuclear cells (SMCs). Using both TLR7agonist and TLR7silencing lentivirus, we found stimulation of TLR7decreased platelet counts and increased levels of platelet-associated IgG (PAIgG) in ITP mice, which correlates TLR7with platelet destruction by autoantibodies. Levels of serum BAFF increased significantly in ITP mice and stimulation of TLR7promoted secretion of BAFF. Among the three BAFF receptors, only BAFF receptor (BAFF-R) increased in ITP mice. However, activation of TLR7showed no effect on the expression of BAFF receptors. These findings indicate that upregulation of TLR7may augment BAFF secretion by APC and through ligation of BAFF-R promote autoreactive B cell survival and thus anti-platelet autoantibody production. The pathway of TLR7/BAFF/BAFF-R provides us with an explanation of how activation of APC affects autoantibody production by B cells in ITP and thus might provide a reasonable therapeutic strategy for ITP.