An Experimental Study On The Regulation Of TLR9/IDO Signaling Pathway By Thymosin α1 For Immunotherapy Of Aplastic Anemia

Objective: Aplastic anemia behaves as an immune-mediated disease characterized by pancytopenia and hypoplastic bone marrow. Autologous T lymphocytes are thought to cause bone marrow failure by immune-mediated excessive apoptosis of stem and progenitor cells. In the bone marrow, bone marrow-derived mesenchymal stem cells (MSCs) are considered to be immune-gatekeeper cells, monitoring the cells entering and exiting the bone marrow, and can function as immune suppressors, which is relevant in the maintenance of hematopoietic homeostasis within the bone marrow. Immunosuppression of MSC is mediated by indoleamine 2,3-dioxygenase (IDO) which is expressed and activated by TLR9 signaling pathway. Immunosuppressive function mediated by MSCs is deficient in patients with AA, which leds to an increased response of autoreactive T cells resulting in apoptosis of hematopoietic stem cells.The purpose of this experiment was to study the regulation of thymosinα1 (Tα1) on immunosuppression of bone marrow MSCs through TLR9/IDO signaling pathway and its improvement of hematopoietic function for immunomodulatory therapy of AA. Methods: 1. Bone marrow T cell subsets and phenotype of MSCs from children with AA and normal individuals were measured by using flow cytometry (FCM). 2. Expressions of TLR9 and IDO of MSCs cocultured with or without Tα1 were determined by reverse transcription-polymerase chain reaction (RT-PCR). 3. Inhibition of PHA-activated T cell proliferation by MSCs cocultured with or without Tα1 for 18hrs was detected by using MTT assay. Results: 1. CD4+/CD8+ ratio (0.93±0.41) of bone marrow T cells in children with AA was significantly lower than that in normal individuals (1.42±0.5); CD8+/CD38+ ratio (0.924±0.04) of bone marrow T cells in children with AA was significantly higher than that in normal individuals (0.631±0.056). 2. Bone marrow MSC from normal individual did not express TLR9 and IDO mRNAs. Bone marrow MSC from children with AA obviously expressed TLR9, but not IDO; AA MSCs treated with Tα1 downregulated TLR9 expression but upregulated IDO expression with concentration and time-dependent ways. 3. The inhibition of MSCs in children with AA on PHA-activated T cell proliferation (21.3825±12.34%) was significantly lower than that in normal individual (62.72±17.79%, P<0.05), while AA MSCs treated with Tα1 for 18hrs exhibited a stronger inhibition (42.83±16.54%, P<0.05). Conclusion: 1. T-lymphocyte subsets from bone marrow were abnormal, CD4+/CD8+ ratio reduced and CD8+/CD38+ ratio increased in children with AA, suggesting that activation of autoreactive T cells or CTL within bone marrow may be related to the biological basis for immuno-pathogenetic mechanism of AA. 2. The immunosuppressive effect of bone marrow MSCs on PHA-stimulated T-lympfocyte proliferation and activation decreased significantly in children with AA. 3. The immunosuppression mediated by MSC could be improved by Tα1 through upregulation of IDO expression via TLR9-dependent signaling pathway. As an immunomodulator, Tα1 can improve the hematopoietic function of bone marrow in children with AA.