Elevated Profile Of Th9 Cells And Correlations With Th1 And Th17 Cells In Patients With Immune Thrombocytopenia

Background:Immune thrombocytopenia (ITP) is an autoimmune disease characterized by low platelet count with normal bone marrow and undefined causes of thrombocytopenia. The pathophysiology of ITP is unclear and becoming more complex. A large number of studies have shown that the immune intolerance of platelet antigen can cause the destruction of platelets, and the cytotoxic T cells (CTL) may lead to the ruptureof the platelets. The disorder of megakaryocyte apoptosis can cause the immaturity of platelets, which is another important mechanism in the process of ITP. Besides the humoral immune response, cellular immunity plays an important role in the process of ITP. It is well established that T helper (Th) cells are crucial in modulating the immune system's direction towards ITP. Th1 cells can activate macrophages, and secrete IFN-?and IL-2. Th1 cells are regarded as a key role in the pathogenesis of ITP, and the Th1/Th2 imbalance can lead to the differentiation of B cells and the production of antibodies. Another subset of T cells called Th17 cells was identified to be involved in inflammatory and autoimmune disease, including experimental autoimmune encephalitis, inflammatory bowel disease and arthritis. More recently, a new subset of T cells, characterized by expression of high amounts of IL-9 had been described as Th9 cells.IL-9 is believed to be a new therapeutic target for the regulation of inflammatory and high reactivity in some autoimmune diseases. The abnormal activation of Th9 and the abnormal secretion of IL-9 may be related to the pathogenesis of ITP. IL-9 is regulated by a variety of cytokines and transcription factors, including PU.1 (encoded by Spil), STAT6, Batf, GATA-3 and IRF4, etc. Regarded as the transcription factor of Th9 cells, PU.1 is required for the differentiation and functional activation of Th9 cells. PU.1 can bind directly to the IL-9 promoter to trigger specific chromatin modifications. Therefore, PU.1 plays an important role in regulating the generation of IL-9. Histone modifications associated with the phenotype of Th9 have been shown to be dependent on PU.1, suggesting that PU.1 may be involved in the occurrence and development of ITP through regulation of Th9 differentiation.Objective:In this study, we were intended to dectect the role of Th9 in the pathogenesis of ITP. In order to understand the function of Th9 in ITP, this study evaluated the expression of Th9 in ITP patients, and correlated its levels to Thl and Th17 cells.Methods:ITP patients and control donors:Thirty-four ITP patients (15men and 19 women, age rangel8�56 years) were enrolled in this study in the Qilu Hospital of Shandong University. All of the have been diagnosed as IPT according to the ?The American Society of Hematology 2011 evidence-based practice guideline for immune thrombocytopenia?. We also recruited thirty-four healthy adults (13 men and 21 women, aged 26�63 years) as controls. None of these subjects have been treated with immunosuppressant like glucocorticoid for at least 6 months before sampling, or have autoimmune disease, diabetes, cardiovascular disease, cancer and other diseases. The percentages of Thl, Th17 and Th9 cells in PBMCs in ITP patients and control donors group were detected by flow cytometry analysis. Th9 cells were identified as CD3+CD8--IL-9+while the Thl and the Th17 cells were identified as CD3+CD8"-INF-?+, CD3+CD8--IL-17+ respectively. We examined the levels of INF-y, IL-17 and IL-9 through ELISA. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the expression levels of IL-9 mRNA and PU.1mRNA.Results:The percentage of CD3+CD8--IL-9+cells was higher in ITP patients (1.13�0.32%), compared to healthy controls (0.29�0.08%). Interestingly, we didn't detect the concentration of IL-9 in serum both in ITP patients group and healthy group (below the detected level). In order to further understand the role of Th9 in the process and development of ITP, we mastered the mRNA expression of IL-9 and its coding gene PU.1. We can find that the levels of IL-9 mRNA and PU.1 mRNA in ITP patients were almost twice times than they were in healthy controls.The percentage of Th17 cells (CD3+CD8--IL-17+) in ITP patients(1.84�0.43%) differed significantly from healthy controls (1.21�0.38%), while the level of IL-17 in ITP patients'plasma had no difference compared with the healthy controls (below the detected level). The percentage of Th17 cells had a strong positive correlation with the percentage of Thl cells (r=0.6213, p=0.0087). There was a less positive correlation between the percentage of Th9 cells and the percentage of Thl (r=0.4640, p=0.0258), compared with the strong correlation between the percentage of Th17 cells and the percentage of Thl (r=0.5614, p=0.0192).Conclusion:In the peripheral blood of ITP patients, the percentage of Th9 cells and the transcription factor PU.1 were significant higher than they in control group, which indicated that Th9 cells might take an important participant in the pathogenesis of ITP. Th9 cells could coordinate the function of Thl cells in the pathogenesis and development of ITP, cooperating with Th17 cell. As a regulating factor, PU.1 could provide a possibility for the treatment of ITP as a gene target.