315.5: Mesenchymal Stromal Cell Therapy as Promising Option To Increase Organ Availability and Reduce Graft Failure

Abstract

Introduction: As an immunosuppressive cell, myeloid-derived suppressor cells (MDSC) has been reported to play a positive role in kidney transplantation. ATF/cyclic AMP-responsive element-binding (CREB), as a transcription factor, has been reported in macrophages with PGE2 addition. Hypermethylated MDSC induced by PGE2 are also myeloid-origin cells.There have been no reports on whether CREB also plays a regulatory role in this differentiation process. Method: Bone marrow (BM) cells of C57BL/6 were used to induced MDSC with GM-CSF (20ng/mL) and PGE2(1μg/mL) or with GM-CSF (20ng/mL) and EP1/2/4 antagonists. The expression of CREB, DUSP2 and MYD88 were assessed. The methylation level of their promoter region was measured using BSPCR. CREB inhibitor was used to study its effects. Results: In hypermethylated MDSC induced by GM-CSF and PGE2 and bone marrow cells induced by GM-CSF and EP1/2/4 antagonists, CREB was found in both of them. Meanwhile, two other proteins, DUSP2 and MYD88, which had been reported to be elevated in LPS-stimulated macrophages and did not decrease after the addition of PGE2, were also increased in MDSC compared with cultured BM cells. After adding CREB inhibitor (KG-501) to the hypermethylated MDSC culture system, we tested the expression of these two proteins again. The expression levels of both proteins were reduced. The methylation level of the promoter region of the two was higher than that of the group without CREB inhibitor. The DNA methyltransferase 3 Alpha (Dnmt3a) was not found any difference with or without inhibitor. Conclusion:In MDSC induced by GM-CSF and PGE2, CREB is involved in the regulation of DUSP2 and MYD88, possibly by competing with Dnmt3a on DNA binding in the regulation of promoter methylation. This is the first study focusing on the relation between CREB and DNA methylation by Dnmt3a.