The Effects Of Tc17、Th22、Treg/Th17Subsets In Primary Immune Thrombocytopenia

Study of Tc17subset frequency in patients with immune thrombocytopeniaPrimary 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. Immune thrombocytopenia (ITP) is characterized by a low platelet count, which is the result of both increased platelet destruction and (or) insufficient platelet production. Mechanism of abnormal cellular 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. T-lymphocyte abnormalities are considered important in ITP. However, the mechanism of cellular abnormal immunity in ITP is still not clear.Studies show that T cells with potential autoreactive activity can be found in healthy adult individuals. Many cellular mechanisms of immune modulation have been described, such as the T helper1(Th1) bias, the decreased number or defective suppressive function of regulatory T cells, and the platelet destruction by cytotoxicity T lymphocytes (CTL), also there is research about new animal model that shows CTL mediated thrombocytopenia.Th17is a novel Th subset characterized by the production of IL-17. Th17has been shown to play a crucial role in the induction of autoimmune diseases including rheumatoid arthritis, experimental autoimmune encephalomyelitis (EAE), and allergen-specific responses. In our previous works, we detected the Th17level in peripheral blood of ITP patients and controls by flow cytometry through intracellular cytokines analysis and demonstrated for the first time that Th17was elevated in ITP patients.T-bet is a new transcription factors belonging to T-box family, which expresses selectively in Thl cells. As a specific transcription factors of Thl, it induces the production of IFN-γ, and plays a decisive role in differentiation of Thl cells. A study showed that CD8+T cells that lack T-bet are shunted into an IL-17-producing phenotype. IL-17-secreting CD8+T cells can also be observed in mice deficient in T-bet, where they appear to play a role in allograft rejection. More recently, a new subset of IL-17-secreting CD8+effector cells, termed Tc17, have received marginal attention. One study suggested that IL-17-secreting CD8+T cells (Tc17) may represent a subset that is distinct from IFN-γ-secreting CD8+T cells (Tc1).The involvement of Tc17in various conditions, such as infection, tumor, and autoimmune diseases, has been reported, and showed that IL-17, tumor necrosis factor (TNF), and IFN-g derived from the Tc17cells all played a role in an enhanced recruitment of T cells, neutrophils, and macrophages. However, unlike Th17, Tc17cells have received only marginal attention. And, the characteristics of Tc17and the correlation between Tc17and Th17in ITP patients have not been systematically investigated.To further investigate the possible role of Tc17cells in the pathogenesis of ITP, we measured the levels of Tc17cells and correlated their levels to Th17cells and evaluated their clinical relevance. Objective:◆To detect Tc17level in ITP patients, and analyze the relationship with Th17cells.◆Dexamethasone inhibits peripheral blood PBMC of ITP patients to express IL-17in vitro, and we detect Tc17expression changes.◆To study immune tolerance of Tc17by examining its level in ITP patients.Materials and Methods:◆Selection of cases and normal controls:46cases of newly diagnosed ITP patients. Normal controls come from34healthy volunteers.◆IL-17-producing CD3+CD8+cells as Tc17and IL-17-producing CD3+CD8-cells as Th17were evaluated by flow cytometry and expressed as a percentage of the total CD3+cells.◆Separate mononuclear cells (PBMCs) from peripheral blood of ITP patients, use Dex to culture3d. Tc17cells percentage of the total CD3+cells was detected after culture.◆Specific anti-platelet GPⅡ b/Ⅲa and/or GPⅠb/Ⅸ autoantibodies was measured by modified monoclonal antibody specific immobilization of platelet antigens (MAIPA).Results:◆IL-17intracellular expression in T cells of ITP patients◆Compared with normal controls, percentage of Th17cells was significantly increased in T cells of ITP patients (0.79±0.69%vs0.39±0.33%, P<0.001).◆As for Th17cells, a statistical difference was confirmed between ITP patients and controls (2.10±1.33%vs1.40±0.90%, P<0.01)◆Correlation between Tc17and Th17◆Frequencies of Tc17cells and Th17cells were positively correlated in healthy controls (r=0.7239, P<0.0001), but not in ITP patients (r=-0.0817, P=0.590).◆Tc17Positively correlated with CD8/CD4ratio or with CD8±cells in ITP◆Ratio of CD4/CD8was decreased in ITP patients (1.47±0.91) compared to healthy controls (2.10±0.82, P=0.029), and percentage of CD8was elevated in ITP (30.97±12.19%) compared with control group (23.99±5.92%, P=0.025). ◆Tc17and CD8/CD4ratios were positively related (P=0.001).Tc17and CD8+cells were also positively related in ITP patients (P=0.006).◆Marginally elevated Tc17in autoantibodies-negative ITP patients◆The level of Tc17cells was marginally elevated in patients who had negative MAIPA test(1.15±1.09%) compared to those with positive MAIPA test (0.57±0.25%, P=0.07).◆Impacts of Dexamethasone(Dex) on Tc17in PBMCs of ITP patients◆Tc17percentage had a decreasing tendency with the increase of Dex concentration in culture media.Conclusions:◆Tc17subset percentage in peripheral blood of ITP patients was increased.◆Tc17was positive related to Th17cells in control peripheral blood, while no relationship with Th17subset in ITP patients.◆Dex inhibited Tc17cells frequency in PBMCs of ITP patients in vitro. In summary, Tc17cells subset of ITP patients is decreased, and cellular immuneabnormal is one of the pathogenic mechanisms of ITP. Dex may decrease Tc17frequency of ITP patients. Tc17subset may play an important role in the onset of ITP,providing blockade of IL-17may be a reasonable therapeutic strategy for ITP. Study of Th22subset frequency in patients with immune thrombocytopeniaPrimary 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.Recent years, in the continuous study of cellular immunity, some new helper T cells subsets have been found. Such as Th17cells subset, Th9cells subset, all play an important role in some autoimmune diseases and allergic inflammation by variety pathways.Biological characteristics of Th22cells could be detected on CFSE marked peripheral T cells, langerhans cells (LCs) of the marginal zone and dendritic cells (DCs). By flow cytometry analysis of T cells proliferation, it was found that LCs could induce initial CD4+T cells to produce IL-22, DCs cells’ effect were weaker than LCs, but still can induce IL-22production, showing that both LCs and DCs can induce the initial CD4+T cells differentiating to Th22cells subset, with little IL-17A production in this proceed. Furthermore, differentiation of Th22cells subset may be partially dependent on the regulation of IL-6and TNF-α. Th22cells subset is a stable and mature T cells subset, memory Th22cells would not differentiate to other subpopulations. At present, the mechanism of Th22cells regulation is not completely clear. Th22cells regulation is similar to the T cells. They mainly secret IL-22cytokines, IL-1β and TNF-γ can enhance IL-22receptor molecule expression pathway, especially IL-22R1and STAT-3pathways. If the body is infected by virus, bacteria or other pathogens, Th22cells and Th17cells could play an important role in the pathogenesis of some autoimmune diseases.STAT3is a member of signal transduction and activation of transcription (Signal Transducer and Activator of Transcription, STAT) protein family. STAT family is a group of related proteins can be activated by different cytokine receptors, and serves as a carrier in the process of cytokine receptors interaction, to keep intrinsic specificity of signal transfer intracellular. Thl7cells differentiation also relies on activation of STAT-3and RORC, and is regulated by various cytokines, including IL-6, IL-1β, transforming growth factor β1(TGF-p1), IL-23A, and autocrine IL-21.IL-22is a cytokine secreted by Th22cells, induces STAT signaling pathway activation in various kinds of cells. Previous research found that STAT-1, STAT-3and STAT-5, could be activated by IL-22. Later research showed that, the rat hepatoma H4IIE cell line with surface receptor conjunction to IL-22rapidly induces JAK1and TYK2activation, and then lead to phosphorylation of STAT-1, STAT-3and STAT-5. IL-22is involved in the pathogenesis of various human certain autoimmune diseases, such as rheumatoid arthritis (RA) and Crohn’s disease, etc. So far no report shows Th22cells subset (CD4+, IFN-γ-IL-17-IL-22+T cells) and Th17cells subset in patients with ITP.In order to study the possible role of Th22subset in the pathogenesis of ITP patients, we detected Th22cells, Thl7cells, Thl cells, plasma IL-22levels, and relative quantification of transcription factor STAT-3and RORC mRNA expression, and evaluated their interrelationship in ITP.Objective:◆To detect Th22frequency and plasma IL-22level in ITP patients, and analyze the relationship among Th22, Th17and Thl cells.◆Analyze relative quantification of transcription factor STAT-3and RORC mRNA expression in PBMCs of ITP.◆To evaluate the effect of Th22subset in ITP patients.Materials and Methods:◆Selection of cases and normal controls:44cases of newly diagnosed ITP patients. Normal controls come from36healthy volunteers.◆Th22, Th17and Thl cells frequencies in PBMCs are evaluated by flow cytometry.◆Enzyme-linked immunoadsorbent assay (ELISA) is used to detect plasma IL-22level.◆Realtime fluorescence quantitative PCR is used to detect STAT-3and RORC mRNA expression.Results:◆Th22cells frequency in peripheral blood of ITP patients Compared with normal controls (0.73%;0.24-1.56%), frequency of Th22cells was significantly increased in T cells of ITP patients (1.19%;0.14-9.51%, P<0.0001).◆IL-22cytokine level in plasma of ITP Compared with normal controls(9.98±1.06pg/ml), IL-22level was significantly increased in ITP patients (13.39±8.33pg/ml; P<0.0001).◆Relationship between Th22subset and plasma IL-22level In ITP peripheral blood, frequency of Th22cells and plasma IL-22level was positively correlated(r=0.8457, P<0.001), not in healthy controls group(r=0.2253, P=0.3014).◆Relationship between Th22and Thl7, Th22and Thl in ITP patients Th22and Th17cells were positively related (r=0.7678, P<0.0001), Th22and Thl were also positively related in ITP patients (r=0.6056, P=0.0047).◆STAT-3and RORC mRNA expression in PBMCs of ITP Compared with controls (1.584;1.057-1.776), STAT-3mRNA expression in ITP patients (1.644;1.164-2.189; P<0.05) increased signaficantly; RORC mRNA level of ITP patients (2.502;1.885-2.846) also significantly increased compared with controls (2.208;1.381-2.886; P<0.05)◆Th22subset elevated in autoantibodies-negative ITP patients Compared to those with positive MAIPA test(0.83;0.23-1.24), Th22cells was signaficantly elevated in patients who had negative MAIPA test(2.35; range,0.39-9.51; P=0.013).Conclusions:◆Th22subset elevated in peripheral blood of ITP patients.◆Plasma IL-22level was increased, and positively related to Th22cells in ITP patients.◆STAT-3and RORC mRNA expression was increased in peripheral blood of ITP patients.In summary, Th22cells frequency and IL-22level in plasma of ITP patients are increased, while relative transcription factors expressions were significantly elevated, suggesting cellular immune abnormal in which Th22cells take part is one of the important pathogenic mechanisms of ITP. Measures focus on Th22cells can provide the treatment of ITP with a new method. Neutralizations of IL-17A and IL-21Regulate Treg/Th17Imbalance via Th17-associated Signaling Pathway in Immune ThrombocytopeniaVariety 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.Treg cells, characterized by expression Foxp3in the nuclei, are functionally immunosuppressive subset of T cells, and play pivotal roles in induction T-cell tolerance. Many autoimmune diseases such as rheumatoid arthritis, Type Ⅰ diabetes and multiple sclerosis now are associated with peripheral Treg deficiencies. Many studies demonstrated that the patients with active ITP have functional defects in Tregs which contribute to breakdown of self-tolerance in ITP. Our previous studies have also observed the functional and quantitative abnormity of Treg in ITP patients.Th17cells are characterized as preferential producers of IL-17A (also known as IL-17), IL-17F, IL-21, IL-22, and IL-26in human. The autocrine activity of IL-21, activation of STAT3, and induction of the orphan nuclear receptor ROR γt are the key regulators of Th17-cell lineage differentiation. Th17cells and their effector cytokines are increasingly being recognized as key players in inflammation, autoimmunity and allergic reactions. Th17cells have been shown to play a crucial role in the induction of autoimmune diseases, including rheumatoid arthritis, experimental autoimmune encephalomyelitis (EAE), and allergen-specific responses. In our previous work, we determined the number of Th17cells in the peripheral blood of ITP patients and healthy controls by flow cytometry through intracellular cytokine analysis, and demonstrated for the first time that Th17cells are elevated in ITP patients. Because of the involvement of abnormal Th17and Tregs in the development and progression of ITP, regulating the Th17/Treg imbalance by blocking crucially effective cytokines would be a novel therapeutic strategy for ITP.Recently, IL-17A and IL-21has been shown to be expressed in higher levels in peripheral of ITP patients. IL-21has positive relationship with Th17subset. These studies raise the possibility that IL-17A and IL-21play a role in ITP. However, the influences of IL-17A and IL-21in ITP patients remain unknown.Objective:In this study, the proliferation, apoptosis and effects on Th17and Treg cells of blocking IL-21and IL-17A in ITP were examined in vitro to determine whether IL-21and IL-17A contribute to ITP pathologic processes. Use the ITP patients and control groups to detect STAT1, STAT3, STAT5, and RORC expression of their PBMCs after culture, to explore the role and mechanism of IL-17A and IL-21cytokines in the pathogenesis of ITP.Materials and Methods:◆Peripheral blood mononuclear cells (PBMCs) of25ITP patients and21healthy controls, CD3+T cells of29ITP patients and24healthy controls were isolated.◆4experimental groups (incubated with IL-17, IL-21, anti-IL-17A, anti-IL-21) and control group were set for72hours incubation in vitro. Each group of PBMCs was incubated with IL-2and all CD3+T cells were treated with anti-CD3/CD28Abs.◆After72hours incubation, proliferation was assessed using CCK8assay.◆Apoptosis ratios, percentages of Tregs and Th17cells were detected by flow cytometry.◆In supernatant, IL-17A cytokine response to the effects of IL-21, and IL-21response to the effects of IL-17A were detected by ELISA.◆RORC, STAT-1, STAT-3and STAT-5mRNA expressions were detected by QT-PCR.Results:◆In ITP, PBMCs were cultured with IL-17A or IL-21:a. we found that Th17cells percentage in PBMCs was increases by IL-17A or IL-21 culture(2.09±2.18%or1.70±1.20%vs1.57±1.53%).b. Tregs percentage was decreases by IL-17A or IL-21culture(5.69±3.01%or5.69±2.09%vs6.19±2.76%).c. Apoptosis ratio was decreased(5.24±4.39%or5.05±4.59%vs6.48±5.59%).◆No similar results was found in healthy controls.◆Proliferation of each group showed no significant difference.◆Interestingly, after CD3+T cells were incubated in vitro, no Th17cells were detected by flow cytometry.◆We also found that nTregs were decreased in ITP patients, and that iTregs from ITP patients were elevated to the same level of healthy controls after PBMCs being incubated72hours with IL-2(6.25±3.39%vs5.88±2.05%, P>0.05) or CD3+T cells being incubated72hours with CD3/CD28mAb(6.25±3.39%vs5.88±2.05%, P>0.05).◆IL-21drives IL-17A cytokine in ITP patients.◆IL-17A enhanced STAT1, STAT3but not RORC or STAT-5in ITP PBMCs, while IL-21upward modulated transcripts of STAT-1, STAT-3, STAT-5, RORC mRNA compared with control group.◆IL-17A mAb down-regulated RORC, STAT-1mRNA, while up-regulates STAT-3and STAT-5mRNA. IL-21mAb increased RORC, STAT-3but decreased STAT-1mRNA.Conclusions:◆Our data provided the first evidence of IL-17A and IL-21-induced Th17re-differentiation in PBMCs and inhibiting Tregs re-differentiation in PBMCs and CD3+T cells as well as modulate IL-17A production, via Th17-associated cytokine signaling pathway(STAT-3, STAT-5, RORC) and Thl-associated cytokine signaling pathway(STAT-1) in vitro.◆Tregs or Th17cells may convert from other cell subsets in the existence of cytokines.◆This study also implies the importance of other cells except for CD3+T cells in PBMCs for Th17differentiation, and anti-CD3/CD28Abs or IL-2might elevate Tregs in vitro more obviously in ITP than in healthy control.In summary, Treg/Thl7cells may play a sensitive role in the pathogenesis of ITP, and redundant IL-17A and IL-21not only factors in T cells disorder but also highlights the potential value of IL-17A and IL-21blockade as a novel therapeutic target for human ITP.