Influences of miRNA-155/PU.1 Signaling Pathway Blockade on Rat Bone Marrow-Derived Dendritic Cell Maturation and Transplantation Immunity_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

 XUXing-wei ¹ , DINGBo-wen ² , ZHUChuan-rong ³ , ZHENGPeng ¹ , FENGTao ¹ ,  JIWu ¹

1. School of Medicine, Nanjing University, Research Institute of General Surgery, Jinling Hospital, Nanjing 210002, Jiangsu Province, China;
2. Department of General Surgery, Xuzhou Chinese Medicine Hospital, Xuzhou 221000, Jiangsu Province, China;
3. Department of General Surgery, Huaian First People's Hospital, Huaian 223001, Jiangsu Province, China;

Corresponding author：

JIWu, Email: jiwusky@126.com

Keywords：

Dendritic cell; miRNA-155/PU.1; Immune tolerance; Intestinal transplantation

DOI：

10.7507/1007-9424.20140039

Video：

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Objective To explore the influence of miRNA-155/PU.1 signaling pathway blockade on bone marrow-derived dendritic cells (DCs) maturation and immune function of rat small intestinal transplantation. Methods The DCs were induced by adherent culture.The critical transcription factor gene PU.1 was designed and PU.1 siRNA was synthe-sized.The DCs were transfected by liposome transfection and a pair of PU.1 siRNA was screened according to the high silencing efficiency.The expressions of DCs surface markers CD80, CD86, and MHC-Ⅱamong three groups (PU.1 silent group, negative control group, and control group) were analyzed by flow cytometry.The IL-10 and IL-12p70 secretion levels in the supernatant were tested by ELISA method.The allogeneic T lymphocyte proliferation was tested by mixed lymphocyte reaction.The transfected cells were intravenously injected into the recipient rat on day 7 before intestinal transplantation.The survival conditions as well as pathological changes were observed in each group recipients. Results ①The surface molecules CD80, CD86, and MHC-Ⅱin the PU.1 silent group were (27.0±5.6)%, (23.6±4.8)%, and (36.8±6.8)%, respectively; versus (74.0±9.4)%, (76.5±8.7)%, and (87.8±11.3)% in the negative control group, respectively, which were significantly lower in former and showing an in creased trend (P < 0.05).②Compared with the negative control group, IL-10 secretion level was significantly increased (P < 0.05), IL-12p70 secretion level significantly decreased (P < 0.05) in the PU.1 silent group.③The proliferation of T lymphocytes in the PU.1 silent group was significantly lower than that in the negative control group (P < 0.05).④When the transfected DCs were injected into the intestinal transplantation rats on day 7 before operation, the survival time was (14.3±3.3) d, (7.8±1.5) d, and (8.0±2.5) d in the PU.1 silent group, negative control group, and control group, respectively, which in the PU.1 silent group were significantly longer than that in the other two groups (P < 0.05), and the graft pathology showed that there were mild intestinal tissue damage, lymphocyte infiltration or villus edema in the PU.1 silent group. Conclusion miRNA-155/PU.1 signaling pathway blockade could reduce DCs maturation and induce receptor-specific immune tolerance, which are proved both in vivo and in vitro.

Citation： XUXing-wei, DINGBo-wen, ZHUChuan-rong, ZHENGPeng, FENGTao, JIWu. Influences of miRNA-155/PU.1 Signaling Pathway Blockade on Rat Bone Marrow-Derived Dendritic Cell Maturation and Transplantation Immunity. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2014, 21(2): 168-172. doi: 10.7507/1007-9424.20140039 Copy

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13.	Leddin M, Perrod C, Hoogenkamp M, et al.Two distinct auto-re-gulatory loops operate at the PU.1 locus in B cells and myeloid cells[J].Blood, 2011, 117(10):2827-2838.
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15.	Wu L, Liu YJ.Development of dendritic-cell lineages[J].Imm-unity, 2007, 26(6):741-750.
16.	Bharadwaj AS, Agrawal DK.Transcription factors in the control of dendritic cell life cycle[J].Immunol Res, 2007, 37(1):79-96.
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18.	Hamdorf M, Berger A, Schüle S, et al.PKCδ-induced PU.1 phosphorylation promotes hematopoietic stem cell differentiation to dendritic cells[J].Stem Cells, 2011, 29(2):297-306.
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1. Santiago-Schwarz F.Positive and negative regulation of the myeloid dendritic cell lineage[J].J Leukoc Biol, 1999, 66(2):209-216.
2. Carotta S, Dakic A, Wu L, et al.The transcription factor PU.1 controls dendritic cell development and Flt3 cytokine receptor expression in a dose-dependent manner[J].Immunity, 2010, 32(5):583-585.
3. Lutz MB, Suri RM, Niimi M, et al.Immature dendritic cells generated with low doses of GM-CSF in the absence of IL-4 are maturation resistant and prolong allograft survival in vivo[J].Eur J Immunol, 2000, 30(7):1813-1822.
4. Yang XJ, Meng S, Zhu CF, et al.Semi-mature MyD88-silenced bone marrow dendritic cells prolong the allograft survival in a rat model of intestinal transplantation[J].Chin Med J (Engl), 2011, 124(2):268-272.
5. 杨阳, 韩岩, 杨麦贵, 等.调节性CD4⁺CD25⁺T细胞的分离纯化及免疫功能研究[J].中华实验外科杂志, 2009, 26(12):1620-1622.
6. Casiraghi F, Aiello S, Remuzzi G.Transplant tolerance:progress and challenges[J].J Nephrol, 2010, 23(3):263-270.
7. Vosters O, Nève J, De Wit D, et al.Dendritic cells exposed to nacystelyn are refractory to maturation and promote the emergenceof alloreactive regulatory T cells[J].Transplantation, 2003, 75(3):383-389.
8. Mahnke K, Schmitt E, Bonifaz L, et al.Immature, but not inac-tive:the tolerogenic function of immature dendritic cells[J].Immunol Cell Biol, 2002, 80(5):477-483.
9. Lutz MB, Schuler G.Immature, semi-mature and fully mature dendritic cells:which signals induce tolerance or immunity?[J].Trends Immunol, 2002, 23(9):445-449.
10. O'Connell RM, Taganov KD, Boldin MP, et al.MicroRNA-155 is induced during the macrophage inflammatory response[J].Proc Natl Acad Sci USA, 2007, 104(5):1604-1609.
11. Rodriguez A, Vigorito E, Clare S, et al.Requirement of Bic/microRNA-155 for normal immune function[J].Science, 2007, 316(5824):608-611.
12. Vigorito E, Perks KL, Abreu-Goodger C, et al.microRNA-155 regulates the generation of immunoglobulin class-switched plasma cells[J].Immunity, 2007, 27(6):847-859.
13. Leddin M, Perrod C, Hoogenkamp M, et al.Two distinct auto-re-gulatory loops operate at the PU.1 locus in B cells and myeloid cells[J].Blood, 2011, 117(10):2827-2838.
14. Leng RX, Pan HF, Qin WZ, et al.Role of microRNA-155 in autoimmunity[J].Cytokine Growth Factor Rev, 2011, 22(3):141-147.
15. Wu L, Liu YJ.Development of dendritic-cell lineages[J].Imm-unity, 2007, 26(6):741-750.
16. Bharadwaj AS, Agrawal DK.Transcription factors in the control of dendritic cell life cycle[J].Immunol Res, 2007, 37(1):79-96.
17. Carotta S, Dakic A, D'Amico A, et al.The transcription factor PU.1 controls dendritic cell development and Flt3 cytokine receptor expression in a dose-dependent manner[J].Immunity, 2010, 32(5):628-641.
18. Hamdorf M, Berger A, Schüle S, et al.PKCδ-induced PU.1 phosphorylation promotes hematopoietic stem cell differentiation to dendritic cells[J].Stem Cells, 2011, 29(2):297-306.
19. Thompson RC, Herscovitch M, Zhao I, et al.NF-kappaB down-regulates expression of the B-lymphoma marker C D10 through a miR-155/PU.1 pathway[J].J Biol Chem, 2011, 286(3):1675-1682.
20. Boks MA, Kager-Groenland JR, Haasjes MS, et al.IL-10-gener-ated tolerogenic dendritic cells are optimal for functional regulatory T cell induction-a comparative study of human clinical-applicable DC[J].Clin Immunol, 2012, 142(3):332-342.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Influences of miRNA-155/PU.1 Signaling Pathway Blockade on Rat Bone Marrow-Derived Dendritic Cell Maturation and Transplantation Immunity

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