Bone Marrow Derived Mesenchymal Stem Cells Induce The Generation Of Novel Regulatory Dendritic Cells Through Notch Signaling

Background Mesenchymal stem cells (MSCs) can be isolated from many tissues and organs, such as bone marrow, umbilical cord and adipose. Because of their unique biological properties, such as low immunogenicity as well as immunomodulatory functions, MSCs catches a lot of attention in the field of regenerative medicine. MSCs play out their immune regulatory functions possibly through various soluble anti-inflammatory components that they secrete or direct cell-cell contact with other cells. Dendritic cell plays an important role in congenital immunity and acquired immunity. It is also known that the regulatory DC cell plays a significant role in immune system. So studying on the MSCs, which could induce the hematopoietic stem cells to produce the regulatory DC, provides a theoretical basis for stem cell therapy. Our previous study found that MSCs that derived from MEF could affect the production of regulatory DC through secreting soluble substances. However, it is still unknown that whether MSCs derived from bone marrow could affect the production of regulatory DC Without any exogenous cytokines. We also discovered that MSCs derived from bone marrow (bone marrow MSCs, BM-MSCs) expressed a Notch ligand. So the functions of the notch pathway on the regulatory DCs production in the MSC coculture system need further research. This study mainly focused on the role of Notch signaling in the generation of the regulatory DC and its detailed mechanisms.Objective Based on the above research status, we investigated systematically the detailed mechanisms of BM-MSCs induceing hematopoietic stem cells to produce the regulatory DC.First, immunophenotype of regulatory DC, expression of transcription factor and excreted factor were tested.Second, the immune function of DC both in vitro and in vivo and its therapeutic effect in the IBD model was explored.Third, the role of Notch signaling in the generation of the regulatory DC induced by BM-MSCs and its detailed mechanisms ware studied.Methods First, biological properties of BM-MSCs isolated with magnetic beads were analyzed based on ELISA, qRT-PCR and Flow cytometry. Second, BM-MSCs induced the hematopoietic stem cell to produce a novel regulatory cell, sBM-DCs, and their biological properties were studied by Flow cytometry. Transcription factor expression was analyzed by Western blot, qRT-PCR in protein or mRNA level. Cytokine secretion was also detected by ELISA. Third, we used mixed lymphocyte reaction and DTH experiment to analyze the immunomodulatory properties of sBM-DCs both in vitro and vivo and Flow cytometry was used to test the effect of sBM-DCs on T cell activation markers. Fourth, the effect of sBM-DCs on the IBD model was analyzed by HE staining of pathology slices and the molecular biological mechanism by flow cytometry. Fifth, introducing the Notch pathway inhibitor to the coculture system, the biological properties of sBM-DCs was studied by flow cytometry, Western blot and ELISA, mixed lymphocyte reaction was used to analyze the immunomodulatory properties. Sixth, the siRNA silencing technique was used knock down the Notch ligand in the BM-MSCs and these cells are cocultured with HSCs to analyze the difference with sBM-DCs. Seventh, we identified the molecular biological mechanism of Notch pathway using lentiviral vector, Chip and Co-IP. Eighth, the role of epigenetic regulation in the generation of sBM-DCs was also studied using CHIP method.Results Without any exogenous cytokines, the BM-MSCs can induce HSCs to differentiate into a novel regulatory DC, named as sBM-DCs. The sBM-DCs expressed a higher level of CD11b, and a lower level of CD11c, CD40, CD80, CD86, la in flow cytometry. Western blot experiments showed a high level of PU.1, Ikaros, RBP-J, and a relatively lower expression of IRF4, IRF8, Batf3, SpiB, TCF4 and no expression of Relb, TRAF6, c-Maf and MafB in these sBM-DCs. These cells also secrete high levels of IL-10.The sBM-DCs can alleviate immune reaction both in vitro and in vivo. In vitro, these sBM-DCs can significantly reduce the lymphocyte proliferation while suppress the expression of T-cell activation markers, CD44 and CD69. Meanwhile, sBM-DCs can effectively alleviate the DTH reaction in vivo. These sBM-DCs can also reduce inflammation reaction and the secretion of IL-2, TNF-αt in a IBD mouse model, while increase the secretion of IL-10, and the ratio of CD4+ CD8+double-positive regulatory T cells in PBMCs in the same animal model.The Notch signaling plays an important role in the generation of sBM-DCs. After adding the Notch pathway inhibitor DAPT while knocking down the expression of Jagged1, a Notch ligand, the result is similar. CD11b decreased and the level of CD40、CD80、CD86、Ⅰa is increased, accompanied by decreasing of Ikaros、RBP-J and increasing of IRF8、TCF4、TRAF6. The secretion of IL-10 is decreased and the secretion of IL-12 is increased, indicating lost of immunoregulation.Notch signal participated in epigenetic modulation of RBP-J to regulate the expression of IRF8 is fundamental for sBM-DCs generation. Silencing of IRF8, both mRNA and protein level of IL-12 decreased. IRF8 directly controlled the expression of IL-12 by binding to the promoter region of IL-12. Also, knocking down of RBP-J caused the direct loss of IRF8 in mRNA and protein level, showing that IRF8 expression requires the presence of RBP-J. Interference with RBP-J as well as introducing DAPT could not reverse the expression of IRF8. In sBM-DCs, the RBP-J bound more SIRT1 inhibits the expression of IRF8. After introducing DAPT, RBP-J bound less SIRT1 and the inhibition of IRF8 is lost. Notch signaling can also change the histone modification status of IRF8 to regulate its expression.Conclusion First, without any exogenous cytokines, the BM-MSCs can induce HSCs to differentiate into a novel regulatory DC, which has a strong phagocytic ability and low immunogenicity. There is a high expression of PU.1, Ikaros and RBP-J, and a low expression of IRF4, IRF8, Batf3, SpiB and TCF4 in the DCs. Meanwhile, Relb, TRAF6, c-Maf and MafB expressed negatively, while IL-10 expressed a high level.Second, the novel sBM-DCs are equipped with immune function both in vitro and in vivo.Third, the Notch signaling plays an important role in the generation of sBM-DCs and through RBP-J and epigenetic modulation to regulate the expression of IRF8, sustaining the anti-inflammatory status of sBM-DCs.