Abstract

Aplastic anemia(AA) is an unusual hematologic disease and the paradigm of the human bone marrow failure syndromes. Abnormal immunological responses have been found in most aplastic patients. Activated type 1 cytotoxic T cells may be the main contributing factor of pathogenesis. IDO is a rate-limiting enzyme in Tryptophan(Trp) metabolism. The proliferation of Th1 cells is specifically inhibited by the over-expression of IDO, which can degrade the Trp in local environment, hamper the conductioin of the activating signal in T cell and induce immune tolerance. 3,4-DAA is an IDO agonist and can activate IDO. In our experiment, Balb/c mice were irradiated (5.0Gy of 60Co), and then infused with 5×106 lymph node(LN) cells from DBA/2 mice in 4 hours. Blood count was monitored and marrow damage was assessed by histogical study. Concentrations of serum IFN-γ were measured by ELISA. The levels of Tryptophan and kynurenine were evaluated by high performance liquid chromatography (HPLC). CD4+CD25+ T cells in spleen were analyzed by flow cytometry. The level of Foxp3 in CD4+CD25+ T cell was measured by RT-PCR. Irradiation and infusion of LN cells led to rapid development of severe pancytopenia and bone marrow hypoplasia. Bone marrow of affected mice showed lymphocyte infiltration. Serum IFN-γ concentration increased 3.7 fold at d6 post infusion. The recipient mice were divided into 4 different treating groups as follow:

  1. 0.9% Sodium Chloride as control;

  2. Cyclosporin A (CsA) (50ug/g/d ×5d, peritoneal injection);

  3. 3,4-DAA(5mg/d, orally daily);

  4. the combination of CsA and 3,4-DAA.

The effects of CsA, IDO agonist and CsA combined IDO agonist were analyzed at day 6,10,14,21,24 and 28 after LN cells infusion. The white cell and the platelet recovered to near normal, respectively (4.2±0.32)×109, (937±190.47)×1012 at d21 in the combination group. Early stage treatment with CsA can improve periphery blood cells and BM nucleated cells, but long term effect was not remarkable. In contrast, the 3,4-DAA group exhibited slow and gradually enhanced role. Periphery blood cells and BM nucleated cells were improved remarkably in the combination group. The number and function of CD4+CD25+T cells also increased remarkably. In the treatment of AA, abnormal immune response in bone marrow was inhibited by CsA, meanwhile immune tolerance was induced through up-regulating the regulatory T cells(Treg) by 3,4-DAA. In this way, the balance of immune in bone marrow could be reestablished quickly. CsA combined IDO agonist could provide a new strategy for the management of AA.

Author notes

Disclosure: No relevant conflicts of interest to declare.