Rapamycin Can Reduce Apoptosis of CD4+CD25+ Regulatory T Cells from Mouse Model of Severe Aplastic Anemia

Abstract

CD4+CD25+ regulatory T cells (Tregs) are believed to control the development and progression of autoimmunity by suppressing autoreactive T cells and decreased numbers of these cells are associated with impaired immune homeostasis and development of aplastic anemia (AA). We have developed a novel mouse model of severe aplastic anemia (SAA) induced by interferon-gamma in combination with Busulphan, in which excessive apoptotic CD4+CD25+ Treg cells was found in our previous experiments. It might be one of the reasons for reduction of CD4+CD25+ Treg cells in patients with AA. Rapamycin (RAPA) is a macrolide antibiotic produced by streptomyces hygroscopicus and is considered as a potential immunosuppressant in the treatment of AA, while the effect of RAPA on apoptosis of CD4+CD25+ Tregs from patients with AA is not clear. In this study, the SAA model female mice were intraperitoneal injection with RAPA at daily dose of 0.5 mg/kg for 5 days (the RAPA-treated group, n=60), the SAA group (n=60) and the un-treated group (n=60) were as control. All mice selected to set up the SAA model developed the typical clinical and pathological patterns of SAA from day 10 post dosing. Most of them presented bleedings in association with anemia and infections. In order to explore the effect of RAPA on apoptosis of CD4+CD25+ Tregs from the novel mouse SAA model, the mononuclear cells of the peripheral blood and spleen were subjected to assess the intracellular Foxp3 expression in CD4+CD25+ Tregs by flow cytometry (FCM). In the SAA group, the FCM analysis showed down-expression of Foxp3 in CD4+CD25+ Tregs compared with the un-treated group (P<0.05). These results were in accordance with our previous observations. However, after treatment with RAPA, the expression of Foxp3 in CD4+CD25+ Tregs was increased (P<0.05). In addition, after being pured by immunomagnetic beads, the splenic CD4+CD25+ Tregs was subjected to assess apoptosis by FCM and the Akt and Stat3 phosphorylation using western blot. Compared with the un-treated group, increased CD4+CD25+ Tregs apoptosis with increased Akt phosphorylation accompanied by increased Stat3 phosphorylation was found in SAA group (P<0.05, respectively). On the contrary, RAPA-treated group exhibited CD4+CD25+ Tregs with a reduction in apoptosis rate, Akt phosphorylation and Stat3 phosphorylation compared with the SAA group (P<0.05, respectively). These results indicated that RAPA may increase the expression of Foxp3 by down-regulation the levels of Akt and Stat3 phosphorylation in splenic CD4+CD25+ Tregs from the mice model of SAA, through which might RAPA reduce apoptosis of the CD4+CD25+ Tregs from this model. DisclosuresNo relevant conflicts of interest to declare.