The Roles And Mechanisms Of Notch Signaling In Th17/Treg Imbalance In Patients With Immune Thrombocytopenia

Background:Immune thrombocytopenia (ITP) is an autoimmune-mediated disease characterized by immune-dependent accelerated destruction and impaired production of platelets. Although B cell-mediated anti-platelet autoantibodies play an improtant role in it, the accurate pathogenesis of ITP has not been fully elucidated. It was broadly approved that the multi-dysfunctional immunity of T-lymphocyte contributes to the development of the disease. And the roles of activated T helper (Th) cells and cytokines are increasingly receiving attention in the pathophysiology of ITP. Recently, Th17and T regulatory cells (Tregs) have been defined as two more novel distinct CD4+T subsets from Th1and Th2cells. The balance between Th17and Treg cells controls autoimmune response and has been reported to be a key factor in regulating T cell function and the imbalance of Th17/Treg can lead to autoimmune disease. It has been indicated by current researches that the imbalance of Th17/Treg contributes to the development of ITP. The Notch signaling is a pivotal regulator of a variety of cellular functions, including cell proliferation, differentiation and apoptosis. However, the precise role of Notch signaling in Th17/Treg imbalance in ITP is still not clarified.Objective:The purpose of the corrent study was to further testify the imbalance of Th17/Treg in ITP and to explore the roles and mechanisms of Notch signaling in the imbalance of Thl7/Treg in ITP. Our study will further clarify the pathogenesis of ITP and may offer a novel therapeutic target for treatment of ITP.Methords:We selected newly diagnosed patients with ITP enrolled from March2011to May2012in department of Hematology, Qilu Hospital, Shandong University and healthy volunteers as subjects and venous peripheral blood was collected from all the subjects. Peripheral blood mononuclear cells were extracted by Ficoll density gradient centrifugation. Cells in experimental groups were respectively treated with y-Secretase inhibitor DAPT at different concentrations and cells in control group were incubated with equivalent doses of DMSO. After cultured for72hours at a37℃,5%CO2incubator with IL-2stimulation, cells were collected and we examined the percentage of Th17、Treg cells and the alteration of Thl7/Treg ratio in each group by flow cytometry, and the mRNA expression of Notch signaling (Notch1、Hes1and Heyl) and specific transcription factor of Th17and Treg cells (RORyt and Foxp3) was investigated by RT-PCR. Cell proliferation and apoptosis were also respectively observed by the Cell Counting Kit-8and Apoptosis detection Kit. Supernatants were harvested for detection of IL-17、IL-21and IL-10levels in each group by Enzyme linked immunosorbent assay (ELISA).Results:1. Inhibition of Notch signaling by DAPT:After treated with different concentrations of DAPT, there was a reduction of Notchl expression in both ITP patients and controls, but no statistical variation was observed (p>0.05). The expression of Hesl and Heyl was significantly decreased at DAPT groups compared with DMSO group in patients with ITP. And there was a statistical difference between5、10or20μmol/L DAPT group and DMSO group in patients with ITP (p<0.05). Though the mRNA expression of Hesl and Heyl was decreased in healthy controls, no significant difference was found between each DAPT group and DMSO group (p>0.05).2. Inhibition of Notch signaling by DAPT downregulated the enhanced Th17cells and the ratio of Thl7/Treg in patients with ITP:2.1Compared with healthy controls, Th17cells were obviously increased, Treg cells were significantly decreased and the ratio of Th17/Treg was increased in patients with ITP (p<0.05).2.2The percentage of Th17cells and proportion of Th17/Treg in patients with ITP were reduced in a dose-independent manner in each experimental group compared with DMSO group (p<0.05) and decreased to the minimum at20μmol/L DAPT group. No significant difference of Treg cells was found though a slight increase in each experimental group of ITP patients (p>0.05).3. The effect of inhibition of Notch signaling by DAPT on the secretion of Th17/Treg-associated cytokines in ITP patients:Compared with DMSO group, IL-17secretion in each experimental group was significantly reduced in a dose-dependent manner in ITP patients and reduced to the minimum at20μmol/L DAPT group (p<0.05). Compared with DMSO group, no significant difference of the level of IL-21and IL-10was observed in experimental groups of patients with ITP (p>0.05).4. The detection of RORyt and Foxp3mRNA expression after inhibition of Notch signaling by DAPT:4.1Compared with healthy controls, RORyt mRNA expression was markedly increased、Foxp3mRNA expression was significantly decreased and the ratio of RORyt/Foxp3was significantly increased in ITP patients (p<0.05).4.2After treated with DAPT, the mRNA expression of RORyt was significantly reduced in ITP patients in a dose-dependent manner compared with DMSO group (p<0.05), which was reduced to the minimum at20μmol/L DAPT group. No significant difference was observed though a slight increase of Foxp3mRNA expression in experimental groups compared with DMSO group in patients with ITP (p>0.05). There was a slight reduction of RORyt/Foxp3ratio in experimental groups compared with DMSO group in patients with ITP, but no significant difference was found (p>0.05).5. Inhibition of Notch signaling by DAPT induced cell apoptosis:Cells apoptosis was obviously elevated at10μmol/L or20μmol/L DAPT group compared with DMSO group in patients with ITP (p<0.05). Cells apoptosis was higher at20μmol/L DAPT group than DMSO group in healthy controls (p<0.05), and was higher at20μmol/L DAPT group than2.5μmol/L or5μmol/L DAPT group in healthy controls (p<0.05).6. The effect of inhibition of Notch signaling by DAPT on cell proliferation:There was no significant difference of cell proliferation at DAPT groups relative to DMSO group in ITP patients (p>0.05). No remarkable effect of DAPT was found on cell proliferation in healthy controls (p>0.05).Conclusions:1. Th17cells and transcription factor RORyt were increased, and Treg cells and transcription factor Foxp3were decreased, and the ratio of Thl7/Treg and RORyt/Foxp3was increased in ITP patients indicating a crucial role of Thl7/Treg imbalance in the pathogenesis of ITP.2. Inhibition of Notch signaling pathway can significantly decrease Thl7cells, slightly elevate Treg subset and rectify the imbalance of Th17/Treg ratio in patients with ITP.3. Inhibition of Notch signaling pathway can reduce Thl7cytokine IL-17and transcription factor RORyt, but the alteration of Treg cytokine IL-10and transcription factor Fxop3did not reach statistical significance.4. Inhibition of Notch signaling pathway can induce apoptosis of Peripheral blood mononuclear cells from patients with ITP.5. Inactivation of Notch signaling by DAPT can reverse the enhanced Thl7cells and Thl7/Treg imbalance through downregulation of RORyt mRNA expression in ITP patients. Regulation of balanced network of Thl7/Treg by y-secretase inhibitor may be a novel therapeutic target for prospective treatment of ITP.