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研究生:陳恬萱
研究生(外文):Tien-Hsuan Chen
論文名稱:小鼠骨髓間葉幹細胞介白素-1beta的分泌機制與alpha型防禦素間相關性之探討
論文名稱(外文):Study on the role of alpha-defensin in NLRP3 inflammasome dependent IL-1beta secretion of mesenchymal stem cells
指導教授:江伯倫江伯倫引用關係
指導教授(外文):Bor-Luen Chiang
口試委員:徐立中伍安怡
口試日期:2015-07-10
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:免疫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2015
畢業學年度:103
語文別:英文
論文頁數:75
中文關鍵詞:間葉幹細胞NLRP3發炎複合體IL-1betaalpha型防禦素LPS發炎模式
外文關鍵詞:Mesenchymal stem/stromal cellNLRP3 inflammasomeIL-1betaalpha-defensinLPS inflammation model
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近來研究指出間葉幹細胞(mesenchymal stem cells; MSCs)在發炎環境中扮演著調節者的腳色,針對不同種類的發炎刺激,會引發細胞內完全相反的反應。本研究針對小鼠骨髓間葉幹細胞中介白素-1beta(IL-1beta)的基因表現及細胞內製造途徑進行調查。
在BALB/c小鼠模式中,取其腹腔巨噬細胞(peritoneal macrophage; pM)、骨髓衍生巨噬細胞(bone marrow derived macrophage; BMDM)作為對照組,以LPS (lipopolysaccharide)與第二刺激物 (Alum或ATP)的組合模擬NLRP3發炎複合體活化及IL-1beta製造途徑。實驗結果發現,間葉幹細胞在不受刺激的情況下即製造少量IL-1beta,製造途徑仍然透過NLRP3發炎複合體活化。但是,製造出來的IL-1beta並不如預期在經後轉譯修飾後隨即被釋放出細胞外,而是堆積在間葉幹細胞的細胞質內,表達未知的生理意義。經文獻回顧,我們假設一種抗微生物胜肽(antimicrobial peptide) alpha型防禦素 (alpha-defensin)能在不影響製造途徑的前提下,調控IL-1bera分泌的能力。比諸作為對照組的巨噬細胞,alpha型防禦素的製造在間葉幹細胞內旺盛許多。並且,在使用免疫沉澱(immunoprecipitation)的成果中,我們也看到alpha型防禦素與IL-1beta間具直接相互結合的能力。然而,關於兩者之間關係的直接證據,尚待繼續研究。
總結而言,本篇研究在間葉幹細胞中發現了自發性活化的NLRP3發炎複合體及在該細胞中IL-1beta具有獨特的分布現象。我們進一步假設了alpha型防禦素在發炎環境中的腳色與IL-1beta的分泌相關。本研究的主要貢獻在於報導一個全新且獨特存在於間葉幹細胞中的反應途徑,使我們對於該細胞在發炎環境中的反應有更深一層的認識。

In recent studies, the role of mesenchymal stem/stromal cells (MSCs) has become a modulator of inflammation in response to different inflammatory signals. Among all, we focus on the profile of NLRP3 inflammasome and its main product cytokine IL-1beta.
Secretion and processing pathway of IL-1beta in BALB/c mice thioglycollate-elicited peritoneal macrophages (pMs), bone marrow derived macrophages (BMDMs) and BM-MSCs were investigated at both protein and RNA level after lipopolysaccharide (LPS) and a secondary stimulator (Alum or ATP) treatment. Interestingly, we found the NLRP3-caspase 1-IL-1beta pathway autonomously launched without LPS stimulation in MSCs. Moreover, MSCs maintain this small amount of IL-1beta in their cytosol for unknown physiological purpose.
Since this discovery is apart from our knowledge of secretion right after production, we further postulated that mouse alpha-defensins, cryptdin 1 and cryptdin 4, might play a modulatory role in blocking the release of IL-1beta based on the previous reports and our observation of their larger amount in MSCs compared to pMs. Furthermore, the immunopreicipitation data also supported alpha-defensins would bind with IL-1beta in the cell.
In conclusion, we demonstrated autonomous NLRP3 inflammasome activation in MSCs and a possible new role of alpha-defensins involved in inflammation. We believed that this investigation here might shed light on further understanding the mechanism of IL-1beta release and also therapeutic strategies of high-efficacy MSC-based therapy in autoimmune diseases.


口試委員會審定書 i
誌謝/Acknowledgement ii
中文摘要 iii
Abstract iv
Contents vi
Figure Contents xii
Table Contents xiii

Chapter I Introduction 1
1. Overview of Mesenchymal Stem/Stromal Cells 2
1.1 Characterization of MSCs 2
1.2 Immunoregulatory Activities of MSCs 3
1.3 MSCs in inflammation 4
1.4 MSC-based cellular therapy 4
2. IL-1beta production in inflammation 5
2.1 Characterization of IL-1beta in inflammation 5
2.2 Regulation of IL-1beta production 6
2.3 Caspase-1 independent processing pathway 6
3. Overview of alpha-defensin 7
3.1 Characterization of alpha-defensin 7
3.2 Mechanisms of alpha-defensin bactericidal activity 8
3.3 The role between alpha-defensin and IL-1beta release 9
4. Hypothesis and Specific aims 10
Chapter II Materials and Methods 11
Part1 Materials 12
1. Cell Culture 12
1.1 Animals 12
1.2 Cell culture medium and buffer 12
1.3 MACS cell purification 13
2. Flow cytometry 14
3. Characterization of MSC 14
3.1 MSC differentiation medium 14
3.2 Imobilzer and indicator 15
3.3 T cell proliferation assay 15
4. IL-1beta inducing model 16
5. Immunoblot 16
5.1 Protein extraction and quantification 16
5.2 Gels, buffers, and reagents 16
5.3 Antibodies 17
6. Immunoprecipitation 18
7. Enzyme-linked immunosorbent assay (ELISA) 18
8. Detection of RNA expression 19
8.1 RNA extraction 19
8.2 Reverse transcription-polymerase chain reaction (RT-PCR) 19
8.3 Quantitative real-time polymerase chain reaction (qPCR) 20
9. shRNA knockdown system 21
Part2 Methods 23
1. Cell Culture 23
1.1 Preparation of bone marrow derived MSCs and macrophages (BMDM) 23
1.2 Preparation of peritoneal macrophages (pMs) 23
1.3 CD4+ T cell isolation 24
1.4 General cell culture process 25
1.5 MTT cytotoxicity assay 25
2. Flow cytometry 25
3. Characterization of MSC 26
3.1 MSC differentiation assay 26
3.2 T cell suppression assay 26
4. IL-1beta inducing model 27
5. Immunoblot 27
5.1 Protein extraction 27
5.2 BCA assay 28
5.3 Gel electrophoresis 28
5.4 Transfer and blocking 28
5.5 Detection and stripping 28
6. Immunoprecipitation 29
7. Enzyme-linked immunosorbent assay (ELISA) 29
8. Detection of RNA expression 30
8.1 RNA extraction 30
8.2 Reverse transcription-polymerase chain reaction (RT-PCR) 31
8.3 Quantitative real-time polymerase chain reaction (qPCR) 31
9. shRNA knockdown system 32
9.1 Amplification and isolation of plasmids 32
9.2 293 T transfection 32
9.3 Lentivirus titration 32
9.4 MSC infection 33
10. Statistical analysis 33
Chapter III Results 34
1. Experimental design 35
2. MSCs produced both precursor and mature IL-1beta without LPS stimulation. 36
3. NLRP3 inflammasome expressed and activated in post-translational level without LPS stimulation in MSCs. 37
4. Cryptdins serve as a potential regulator of IL-1beta release in MSCs 38
5. Knockdown of cryptdins in MSCs 39
Chapter IV Discussion 41
1. Regulation of NLRP3 inflammasome in MSCs 42
2. Physiological purpose of IL-1beta accumulation in MSCs 44
3. Regulation of IL-1beta release 46
4. Macrophages from difference source in LPS inflammation model 47
5. Conclusions 48
References 50
Figures 61
Tables 74

1.Luria, E.A., A.F. Panasyuk, and A.Y. Friedenstein, Fibroblast colony formation from monolayer cultures of blood cells. Transfusion, 1971. 11(6): p. 345-9.
2.Uccelli, A., L. Moretta, and V. Pistoia, Mesenchymal stem cells in health and disease. Nat Rev Immunol, 2008. 8(9): p. 726-36.
3.Pittenger, M.F., et al., Multilineage potential of adult human mesenchymal stem cells. Science, 1999. 284(5411): p. 143-7.
4.Lv, F.J., et al., Concise review: the surface markers and identity of human mesenchymal stem cells. Stem Cells, 2014. 32(6): p. 1408-19.
5.Dominici, M., et al., Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006. 8(4): p. 315-7.
6.Boxall, S.A. and E. Jones, Markers for Characterization of Bone Marrow Multipotential Stromal Cells. Stem Cells Int, 2012. 2012.
7.Halfon, S., et al., Markers distinguishing mesenchymal stem cells from fibroblasts are downregulated with passaging. Stem Cells Dev, 2011. 20(1): p. 53-66.
8.Meirelles Lda, S. and N.B. Nardi, Murine marrow-derived mesenchymal stem cell: isolation, in vitro expansion, and characterization. Br J Haematol, 2003. 123(4): p. 702-11.
9.Koide, Y., et al., Two distinct stem cell lineages in murine bone marrow. Stem Cells, 2007. 25(5): p. 1213-21.
10.Anderson, P., et al., CD105 (endoglin)-negative murine mesenchymal stromal cells define a new multipotent subpopulation with distinct differentiation and immunomodulatory capacities. PLoS One, 2013. 8(10): p. e76979.
11.Lin, C.S., et al., Is CD34 truly a negative marker for mesenchymal stromal cells? Cytotherapy, 2012. 14(10): p. 1159-63.
12.Sun, S., et al., Isolation of mouse marrow mesenchymal progenitors by a novel and reliable method. Stem Cells, 2003. 21(5): p. 527-35.
13.Jiang, Y., et al., Pluripotency of mesenchymal stem cells derived from adult marrow. Nature, 2002. 418(6893): p. 41-9.
14.Le Blanc, K. and D. Mougiakakos, Multipotent mesenchymal stromal cells and the innate immune system. Nat Rev Immunol, 2012. 12(5): p. 383-96.
15.Mendes, S.C., C. Robin, and E. Dzierzak, Mesenchymal progenitor cells localize within hematopoietic sites throughout ontogeny. Development, 2005. 132(5): p. 1127-36.
16.Bouffi, C., et al., IL-6-dependent PGE2 secretion by mesenchymal stem cells inhibits local inflammation in experimental arthritis. PLoS One, 2010. 5(12).
17.Nemeth, K., et al., Bone marrow stromal cells use TGF-beta to suppress allergic responses in a mouse model of ragweed-induced asthma. Proc Natl Acad Sci U S A, 2010. 107(12): p. 5652-7.
18.Mougiakakos, D., et al., The impact of inflammatory licensing on heme oxygenase-1-mediated induction of regulatory T cells by human mesenchymal stem cells. Blood, 2011. 117(18): p. 4826-35.
19.Glennie, S., et al., Bone marrow mesenchymal stem cells induce division arrest anergy of activated T cells. Blood, 2005. 105(7): p. 2821-7.
20.Benvenuto, F., et al., Human mesenchymal stem cells promote survival of T cells in a quiescent state. Stem Cells, 2007. 25(7): p. 1753-60.
21.Ghannam, S., et al., Immunosuppression by mesenchymal stem cells: mechanisms and clinical applications. Stem Cell Res Ther, 2010. 1(1): p. 2.
22.Waterman, R.S., et al., A new mesenchymal stem cell (MSC) paradigm: Polarization into a pro-inflammatory MSC1 or an immunosuppressive MSC2 phenotype. PLoS One, 2010. 5(4).
23.Shi, C., et al., Bone marrow mesenchymal stem and progenitor cells induce monocyte emigration in response to circulating toll-like receptor ligands. Immunity, 2011. 34(4): p. 590-601.
24.Le Blanc, K., et al., Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. Lancet, 2008. 371(9624): p. 1579-86.
25.Connick, P., et al., Autologous mesenchymal stem cells for the treatment of secondary progressive multiple sclerosis: an open-label phase 2a proof-of-concept study. Lancet Neurol, 2012. 11(2): p. 150-6.
26.Medzhitov, R., Toll-like receptors and innate immunity. Nat Rev Immunol, 2001. 1(2): p. 135-45.
27.Dinarello, C.A., Immunological and inflammatory functions of the interleukin-1 family. Annu Rev Immunol, 2009. 27: p. 519-50.
28.Maedler, K., et al., Glucose-induced beta cell production of IL-1beta contributes to glucotoxicity in human pancreatic islets. J Clin Invest, 2002. 110(6): p. 851-60.
29.Dinarello, C.A., et al., Interleukin 1 induces interleukin 1. I. Induction of circulating interleukin 1 in rabbits in vivo and in human mononuclear cells in vitro. J Immunol, 1987. 139(6): p. 1902-10.
30.Andrei, C., et al., Phospholipases C and A2 control lysosome-mediated IL-1 beta secretion: Implications for inflammatory processes. Proc Natl Acad Sci U S A, 2004. 101(26): p. 9745-50.
31.Coeshott, C., et al., Converting enzyme-independent release of tumor necrosis factor alpha and IL-1beta from a stimulated human monocytic cell line in the presence of activated neutrophils or purified proteinase 3. Proc Natl Acad Sci U S A, 1999. 96(11): p. 6261-6.
32.Gardella, S., et al., Secretion of bioactive interleukin-1beta by dendritic cells is modulated by interaction with antigen specific T cells. Blood, 2000. 95(12): p. 3809-15.
33.Latz, E., T.S. Xiao, and A. Stutz, Activation and regulation of the inflammasomes. Nat Rev Immunol, 2013. 13(6): p. 397-411.
34.Ouellette, A.J., et al., Mouse Paneth cell defensins: primary structures and antibacterial activities of numerous cryptdin isoforms. Infect Immun, 1994. 62(11): p. 5040-7.
35.Lehrer, R.I., T. Ganz, and M.E. Selsted, Defensins: endogenous antibiotic peptides of animal cells. Cell, 1991. 64(2): p. 229-30.
36.Wilson, C.L., et al., Regulation of intestinal alpha-defensin activation by the metalloproteinase matrilysin in innate host defense. Science, 1999. 286(5437): p. 113-7.
37.Ghosh, D., et al., Paneth cell trypsin is the processing enzyme for human defensin-5. Nat Immunol, 2002. 3(6): p. 583-90.
38.Ouellette, A.J., Paneth cell alpha-defensins in enteric innate immunity. Cell Mol Life Sci, 2011. 68(13): p. 2215-29.
39.de Leeuw, E., et al., Functional interaction of human neutrophil peptide-1 with the cell wall precursor lipid II. FEBS Lett, 2010. 584(8): p. 1543-8.
40.Figueredo, S.M., et al., Anionic amino acids near the pro-alpha-defensin N terminus mediate inhibition of bactericidal activity in mouse pro-cryptdin-4. J Biol Chem, 2009. 284(11): p. 6826-31.
41.Satchell, D.P., et al., Interactions of mouse Paneth cell alpha-defensins and alpha-defensin precursors with membranes. Prosegment inhibition of peptide association with biomimetic membranes. J Biol Chem, 2003. 278(16): p. 13838-46.
42.Ayabe, T., et al., Activation of Paneth cell alpha-defensins in mouse small intestine. J Biol Chem, 2002. 277(7): p. 5219-28.
43.Ayabe, T., et al., Modulation of mouse Paneth cell alpha-defensin secretion by mIKCa1, a Ca2+-activated, intermediate conductance potassium channel. J Biol Chem, 2002. 277(5): p. 3793-800.
44.Ayabe, T., et al., Secretion of microbicidal alpha-defensins by intestinal Paneth cells in response to bacteria. Nat Immunol, 2000. 1(2): p. 113-8.
45.Shi, J., et al., A novel role for defensins in intestinal homeostasis: regulation of IL-1beta secretion. J Immunol, 2007. 179(2): p. 1245-53.
46.Chen, Q., et al., Alarmin HNP-1 promotes pyroptosis and IL-1beta release through different roles of NLRP3 inflammasome via P2X7 in LPS-primed macrophages. Innate Immun, 2014. 20(3): p. 290-300.
47.Perregaux, D.G., et al., Antimicrobial peptides initiate IL-1 beta posttranslational processing: a novel role beyond innate immunity. J Immunol, 2002. 168(6): p. 3024-32.
48.Braun, J., et al., Concerted regulation of CD34 and CD105 accompanies mesenchymal stromal cell derivation from human adventitial stromal cell. Stem Cells Dev, 2013. 22(5): p. 815-27.
49.Chuang, Y.T., et al., Tumor suppressor death-associated protein kinase is required for full IL-1beta production. Blood, 2011. 117(3): p. 960-70.
50.Sokolowska, M., et al., Prostaglandin E2 inhibits NLRP3 inflammasome activation through EP4 receptor and intracellular cyclic AMP in human macrophages. J Immunol, 2015. 194(11): p. 5472-87.
51.Ghonime, M.G., et al., Inflammasome priming by lipopolysaccharide is dependent upon ERK signaling and proteasome function. J Immunol, 2014. 192(8): p. 3881-8.
52.Rossol, M., et al., LPS-induced cytokine production in human monocytes and macrophages. Crit Rev Immunol, 2011. 31(5): p. 379-446.
53.Ding, J., Y.Y. Chou, and T.L. Chang, Defensins in viral infections. J Innate Immun, 2009. 1(5): p. 413-20.
54.Wiens, M.E., et al., Defensins and viral infection: dispelling common misconceptions. PLoS Pathog, 2014. 10(7): p. e1004186.
55.Anderson, J.P., et al., Initial description of the human NLRP3 promoter. Genes Immun, 2008. 9(8): p. 721-6.
56.Clark, E.A., et al., Concise review: MicroRNA function in multipotent mesenchymal stromal cells. Stem Cells, 2014. 32(5): p. 1074-82.
57.Collino, F., et al., MicroRNAs and mesenchymal stem cells. Vitam Horm, 2011. 87: p. 291-320.
58.Chen, T.S., et al., Mesenchymal stem cell secretes microparticles enriched in pre-microRNAs. Nucleic Acids Res, 2010. 38(1): p. 215-24.
59.Bauernfeind, F., et al., NLRP3 inflammasome activity is negatively controlled by miR-223. J Immunol, 2012. 189(8): p. 4175-81.
60.Haneklaus, M., et al., Cutting edge: miR-223 and EBV miR-BART15 regulate the NLRP3 inflammasome and IL-1beta production. J Immunol, 2012. 189(8): p. 3795-9.
61.Medzhitov, R. and T. Horng, Transcriptional control of the inflammatory response. Nat Rev Immunol, 2009. 9(10): p. 692-703.
62.Saccani, S., S. Pantano, and G. Natoli, p38-Dependent marking of inflammatory genes for increased NF-kappa B recruitment. Nat Immunol, 2002. 3(1): p. 69-75.
63.Zaki, M.H., et al., The NLRP3 inflammasome protects against loss of epithelial integrity and mortality during experimental colitis. Immunity, 2010. 32(3): p. 379-91.
64.Agostini, L., et al., NALP3 forms an IL-1beta-processing inflammasome with increased activity in Muckle-Wells autoinflammatory disorder. Immunity, 2004. 20(3): p. 319-25.
65. Sotiropoulou, P.A., et al., Bcl-2 and accelerated DNA repair mediates resistance of hair follicle bulge stem cells to DNA-damage-induced cell death. Nat Cell Biol, 2010. 12(6): p. 572-82.
66.Tatsuta, T., J. Cheng, and J.D. Mountz, Intracellular IL-1beta is an inhibitor of Fas-mediated apoptosis. J Immunol, 1996. 157(9): p. 3949-57.
67.Volarevic, V., et al., Interleukin-1 receptor antagonist (IL-1Ra) and IL-1Ra producing mesenchymal stem cells as modulators of diabetogenesis. Autoimmunity, 2010. 43(4): p. 255-63.
68.Ortiz, L.A., et al., Interleukin 1 receptor antagonist mediates the antiinflammatory and antifibrotic effect of mesenchymal stem cells during lung injury. Proc Natl Acad Sci U S A, 2007. 104(26): p. 11002-7.
69.Arend, W.P., et al., Interleukin-1 receptor antagonist: role in biology. Annu Rev Immunol, 1998. 16: p. 27-55.
70.Hesker, P.R., et al., Genetic loss of murine pyrin, the Familial Mediterranean Fever protein, increases interleukin-1beta levels. PLoS One, 2012. 7(11): p. e51105.
71.Chae, J.J., et al., Gain-of-function Pyrin mutations induce NLRP3 protein-independent interleukin-1beta activation and severe autoinflammation in mice. Immunity, 2011. 34(5): p. 755-68.
72.Wang, C., et al., Characterization of murine macrophages from bone marrow, spleen and peritoneum. BMC Immunol, 2013. 14: p. 6.
73.Leijh, P.C., et al., Effect of thioglycolate on phagocytic and microbicidal activities of peritoneal macrophages. Infect Immun, 1984. 46(2): p. 448-52.

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