T helper 17 (Th17) cells and regulatory T (Treg) cells, along with Th1 and Th2 cells, may contribute to the development of immune thrombocytopenia (ITP). The imbalance of Th17/Treg toward Th17 cells has been shown to play a pivotal role in the peripheral immune response. Notch signaling has been implicated in peripheral T-cell activation and effector cell differentiation. However, the role of Th17/Treg in the pathogenesis of ITP and the effect of Notch signaling on Th17/Treg imbalances remain largely elusive in ITP. In vitro, we treated peripheral blood mononuclear cells (PBMCs) from ITP and healthy controls with γ-secretase inhibitor (DAPT). Th17 cells and Treg cells were measured by flow cytometry and IL-17, IL-21, and IL-10 secretion by enzyme immunoassay technique. The mRNA expression of Ntoch1, Hes1, Hey1, RORγt, and Foxp3 was investigated by RT-PCR. Cell proliferation and apoptosis were determined by the Cell Counting Kit-8 and apoptosis detection kit. We demonstrated that DAPT was effective in inhibiting mRNA expression of Notch signaling molecules. In untreated cultured PBMCs from ITP patients, we observed elevated Th17 cell and IL-21 levels and RORγt mRNA expression, decreased Treg cells and Foxp3 mRNA expression, and an increased ratio of Th17/Treg and RORγt/Foxp3. After inactivating Notch signal by DAPT, Th17 cells and Th17/Treg ratio were dose dependently decreased and accompanied by the reduction of IL-17 in culture supernatants and RORγt mRNA expression in ITP patients. However, no significant difference was found for Treg cells and Foxp3 mRNA expression, RORγt/Foxp3 ratio, and IL-21 and IL-10 levels after DAPT treatment in ITP patients. We also present evidence that the effect of DAPT inhibition on the Th17 cell response was associated with downregulation of RORγt and IL-17 transcription using human in vitro polarization. In conclusion, our findings highlight the importance of Notch signaling in Th17/Treg imbalances in ITP. Inactivation of Notch signaling might be a potential immunoregulatory strategy in ITP patients.
Notch signaling is a critical factor in the imbalance of the Th17/Treg ratio seen in immune thrombocytopenia (ITP). In this study the authors show that inactivation of Notch signaling subsequently downregulates interleukin17 and the transcription factor RORγt, reducing Th17 levels. Therefore, inactivation of Notch signaling might be a potential immunoregulatory strategy for ITP.
Supplementary information The online version of this article (doi:10.1038/labinvest.2014.142) contains supplementary material, which is available to authorized users.
