臺灣博碩士論文加值系統

中文摘要
氣喘是常見的慢性發炎疾病之一，是為呼吸道的過敏反應。當抗原進入到肺部活化 dendritic cells (DC) 後會促使 naïve T cell 分化成 Th2 cell。 Th2 cell 活化後會分泌 IL-4、IL-5、IL-13等 cytokines，使 B cell 活化、促進 eosinophils 的成熟以及黏液的分泌等。間質幹細胞 (Mesenchymal stem cell, MSC) 常被發現於骨髓、臍帶血等間質組織，並且具有多功能分化的潛力。有許多研究指出 MSC 對自體免疫疾病，甚至是呼吸道發炎或過敏反應具有免疫抑制的效果。本篇研究目的主要是探討人類 MSC 在致敏小鼠中的免疫抑制效果，並了解其可能之作用機制。我們使用OVA抗原誘導小鼠氣喘並進行了 MSC 及其培養液的腹腔注射，發現注射 MSC 和培養液的組別中，小鼠肺部嗜酸性球 (eosinophils) 浸潤減少以及OVA刺激脾臟細胞分泌的 IL-5、IL-13、IFN-γ 有下降的趨勢。為了了解 MSC 對於抑制 T cell 增生的能力，我們利用 anti-CD3/CD28 dynabeads 刺激小鼠脾臟細胞並與 MSC 一同培養，結果顯示 MSC 確實可以抑制 T cell 增生且不需透過細胞間的接觸。另外 MSC 也會使 T cell 表面 CD25 表現量下降，而且 MSC 也能有效地抑制 T cell 分泌 IFN-γ，但對 IL-2 並沒有此現象。綜觀結果，我們推論 MSC 可能藉由調節 T cell 的活化作用進而減緩小鼠氣喘的發生，未來可利用 MSC 或其培養液作為對於氣喘的新治療方針。

Abstract
Asthma is a chronic inflammation disease. Allergens activate dendritic cells (DCs) in lung and promote the differentiation of T cells into Th2 cells. These cells secrete cytokines including IL-4, IL-5 and IL-13 that cause B cell activation, eosinophil maturation and mucus hypersecretion, respectively. Mesenchymal stem cells (MSCs) are multipotent stem cells found in varieties of tissues, and have potential to differentiate into several cell types. It has been reported that MSCs have the capability to regulate immune responses in autoimmune diseases. Hence, we hypothesized that MSCs may regulate Th2 immune responses in asthmatic mice. To study the immunomodulatory effects of MSCs in asthma, OVA-sensitized mice were treated with MSCs or culture supernatants. Our results showed that eosinophilia in lung and the levels of IL-5, IL-13 and IFN-γ from OVA-stimulated splenocytes were reduced following MSCs and supernatants treatment. To examine the inhibitory effects of MSCs on T cell proliferation, anti-CD3/CD28 beads-stimulate splenocytes were cocultured with MSCs. The results showed that MSCs decreased the proliferation and CD25 expression of T cells. Additionally, the levels of IFN-γ was significantly reduced, but not IL-2. Taken together, these results suggested that MSCs regulated airway inflammation of asthmatic mice by inhibiting proliferation and activation of T cells. MSCs or their culture supernatants might be applied as a novel strategy for cell-based therapy for asthma.

Contents
指導教授推薦書
論文口試委員會審定書
誌謝 iii
中文摘要 iv
Abstract v
Contents vi
List of tables ix
List of figures x
Chapter 1: Introduction 1
1.1 Asthma 1
1.1.1 Symptoms and prevalence 1
1.1.2 Pathogenesis of asthma 1
1.2 Mesenchymal stem cell 3
1.2.1 Mesenchymal stem cell and tissue regeneration 3
1.2.2 Immunomodulation of MSC 5
1.2.3 MSC suppress airway inflammation and T cell proliferation 5
Chapter 2: Hypothesis and specific aims 7
2.1 Hypothesis 7
2.2 Specific aims 7
Chapter 3: Materials and methods 8
3.1 Animals 8
3.2 MSCs culture and supernatants collection 8
3.3 MSCs characterization 8
3.4 MSCs adipogenic differentiation 9
3.5 Sensitization mice model and mesenchymal stem cells (MSCs) and culture supernatants treatment 9
3.6 Airway hyper-responsiveness (AHR) 10
3.7 Bronchoalveolar lavage fluid (BALF) 10
3.8 OVA-specific antibodies in serum 11
3.9 Splenocytes culture for cytokines detection 12
3.10 The measurement of cytokines by ELISA 13
3.11 RNA extraction from lung tissue 15
3.12 Reverse transcription and real time PCR 15
3.13 MSCs coculture or transwell culture with mouse splenocytes 17
3.14 The surface marker analysis of mouse splenocytes 17
3.15 Statistical analysis 18
Chapter 4: Results 19
4.1 The treatments of MSCs and culture supernatants of MSCs in OVA-sensitized mice 19
4.1.1 There was no effect on AHR with MSCs or culture supernatants treatment in asthmatic mice 19
4.1.2 MSCs and culture supernatants treatment significantly reduced eosinophils infiltration in lung. 19
4.1.3 MSCs or culture supernatants treatments did not change the levels of OVA-specific IgG1, IgG2a, IgE in serum 20
4.1.4 MSCs or culture supernatants treatment significantly decreased IL-13 and IFN-γ production in OVA-stimulated splenocytes 21
4.1.5 MSCs or culture supernatants significantly downregulated the gene expression of IL-5, eotaxin, gob5 and muc5ac in lung 22
4.1.6 The effect of MSCs treatment reducing eosinophilia in lung was better than supernatants 23
4.2 MSCs cocultured or transwell cultured with mouse splenocytes which were stimulated by anti-CD3/CD28 dynabeads 24
4.2.1 MSCs inhibited the proliferation of mouse splenocytes with or without cell contact 24
4.2.2 MSCs did not influence the apoptosis of activated mouse T cells in coculture or transwell culture 25
4.2.3 Expression of CD25 on mouse splenocytes were reduced by MSCs coculture 25
4.2.4 MSCs decreased IFN-γ production of activated mouse T cell significantly both in coculture and transwell culture system 26
Chapter 5: Discussion 27
References 32
Tables 41
Figures 44

List of tables
Table 1. List of primer sequences 41
Table 2. MSCs or culture supernatants had a significant effect on lung eosinophilia 42
Table 3. MSCs or culture supernatants significantly decreased gene expressions for eosinophilia and eosinophils counting in lung 43

List of figures
Figure 1. MSCs or culture supernatants treatment did not change airway hyperresponsiveness (AHR) 44
Figure 2. MSCs and supernatants significantly reduced eosinophils in bronchoalveolar lavage fluid (BALF) 45
Figure 3. MSCs and culture supernatants treatment did not change the levels of OVA-specific antibodies in serum 46
Figure 4. The levels of IL-13 and IFN-γ in OVA-stimulated splenocytes were reduced by MSCs or culture supernatants treatment significantly 47
Figure 5. Gene expression of IL-5 in lung were downregulated by MSCs treatment 48
Figure 6. MSCs and culture supernatants treatment decreased the gene expressions of eotaxins in lung 49
Figure 7. Culture supernatants treatment downregulated the gene expressions of goblet cells and mucus in lung 50
Figure 8. MSCs treatment reduce eosinophilia in lung significantly 51
Figure 9. Proliferation of activated T cells was inhibited by MSCs with or without cell contact 52
Figure 10. MSCs did not influence apoptosis of CD4+ T cells 53
Figure 11. Expression levels of CD25 in mouse T cells was reduced by MSCs in coculture system but not in transwell culture system 54
Figure 12. The levels of IFN-γ were significantly decreased in MSC-presented group 55

References
1. Kudo, M., Y. Ishigatsubo, and I. Aoki, Pathology of asthma. Front. Microbiol., 2013. 4: p. 263.
2. Holgate, S.T., S. Wenzel, D.S. Postma, S.T. Weiss, H. Renz, and P.D. Sly, Asthma. Nat Rev Dis Primers, 2015. 1: p. 15025.
3. Toskala, E. and D.W. Kennedy, Asthma risk factors. Int Forum Allergy Rhinol, 2015. 5 Suppl 1: p. S11-6.
4. Loftus, P.A. and S.K. Wise, Epidemiology of asthma. Curr. Opin. Otolaryngol. Head Neck Surg., 2016. 24(3): p. 245-9.
5. Hammad, H., M. Plantinga, K. Deswarte, P. Pouliot, M.A. Willart, M. Kool, F. Muskens, and B.N. Lambrecht, Inflammatory dendritic cells--not basophils--are necessary and sufficient for induction of Th2 immunity to inhaled house dust mite allergen. J. Exp. Med., 2010. 207(10): p. 2097-111.
6. Holgate, S.T., Innate and adaptive immune responses in asthma. Nat. Med., 2012. 18(5): p. 673-83.
7. Gill, M.A., The role of dendritic cells in asthma. J. Allergy Clin. Immunol., 2012. 129(4): p. 889-901.
8. Caminati, M., D.L. Pham, D. Bagnasco, and G.W. Canonica, Type 2 immunity in asthma. World Allergy Organ J, 2018. 11(1): p. 13.
9. Walford, H.H. and T.A. Doherty, STAT6 and lung inflammation. JAKSTAT, 2013. 2(4): p. e25301.
10. Barnes, P.J., Immunology of asthma and chronic obstructive pulmonary disease. Nat. Rev. Immunol., 2008. 8(3): p. 183-92.
11. Finotto, S., M.F. Neurath, J.N. Glickman, S. Qin, H.A. Lehr, F.H. Green, K. Ackerman, K. Haley, P.R. Galle, S.J. Szabo, J.M. Drazen, G.T. De Sanctis, and L.H. Glimcher, Development of spontaneous airway changes consistent with human asthma in mice lacking T-bet. Science, 2002. 295(5553): p. 336-8.
12. Steinke, J.W. and L. Borish, Th2 cytokines and asthma. Interleukin-4: its role in the pathogenesis of asthma, and targeting it for asthma treatment with interleukin-4 receptor antagonists. Respir. Res., 2001. 2(2): p. 66-70.
13. Fajt, M.L. and S.E. Wenzel, Mast cells, their subtypes, and relation to asthma phenotypes. Ann Am Thorac Soc, 2013. 10 Suppl: p. S158-64.
14. Greenfeder, S., S.P. Umland, F.M. Cuss, R.W. Chapman, and R.W. Egan, Th2 cytokines and asthma. The role of interleukin-5 in allergic eosinophilic disease. Respir. Res., 2001. 2(2): p. 71-9.
15. Rosenberg, H.F., S. Phipps, and P.S. Foster, Eosinophil trafficking in allergy and asthma. J. Allergy Clin. Immunol., 2007. 119(6): p. 1303-10; quiz 1311-2.
16. Zhu, Z., R.J. Homer, Z. Wang, Q. Chen, G.P. Geba, J. Wang, Y. Zhang, and J.A. Elias, Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production. J. Clin. Invest., 1999. 103(6): p. 779-88.
17. Renauld, J.C., New insights into the role of cytokines in asthma. J. Clin. Pathol., 2001. 54(8): p. 577-89.
18. Ullah, I., R.B. Subbarao, and G.J. Rho, Human mesenchymal stem cells - current trends and future prospective. Biosci. Rep., 2015. 35(2).
19. Wei, X., X. Yang, Z.P. Han, F.F. Qu, L. Shao, and Y.F. Shi, Mesenchymal stem cells: a new trend for cell therapy. Acta Pharmacol. Sin., 2013. 34(6): p. 747-54.
20. Pittenger, M.F., A.M. Mackay, S.C. Beck, R.K. Jaiswal, R. Douglas, J.D. Mosca, M.A. Moorman, D.W. Simonetti, S. Craig, and D.R. Marshak, Multilineage potential of adult human mesenchymal stem cells. Science, 1999. 284(5411): p. 143-7.
21. Hass, R., C. Kasper, S. Bohm, and R. Jacobs, Different populations and sources of human mesenchymal stem cells (MSC): A comparison of adult and neonatal tissue-derived MSC. Cell Commun Signal, 2011. 9: p. 12.
22. Dominici, M., K. Le Blanc, I. Mueller, I. Slaper-Cortenbach, F. Marini, D. Krause, R. Deans, A. Keating, D. Prockop, and E. Horwitz, Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy, 2006. 8(4): p. 315-7.
23. De Becker, A. and I.V. Riet, Homing and migration of mesenchymal stromal cells: How to improve the efficacy of cell therapy? World J. Stem Cells, 2016. 8(3): p. 73-87.
24. Dimarino, A.M., A.I. Caplan, and T.L. Bonfield, Mesenchymal stem cells in tissue repair. Front. Immunol., 2013. 4: p. 201.
25. Eggenhofer, E., F. Luk, M.H. Dahlke, and M.J. Hoogduijn, The life and fate of mesenchymal stem cells. Front. Immunol., 2014. 5: p. 148.
26. Gao, P., Y. Zhou, L. Xian, C. Li, T. Xu, B. Plunkett, S.K. Huang, M. Wan, and X. Cao, Functional effects of TGF-beta1 on mesenchymal stem cell mobilization in cockroach allergen-induced asthma. J. Immunol., 2014. 192(10): p. 4560-4570.
27. Wang, Y., X. Chen, W. Cao, and Y. Shi, Plasticity of mesenchymal stem cells in immunomodulation: pathological and therapeutic implications. Nat. Immunol., 2014. 15(11): p. 1009-16.
28. Lee, R.H., A.A. Pulin, M.J. Seo, D.J. Kota, J. Ylostalo, B.L. Larson, L. Semprun-Prieto, P. Delafontaine, and D.J. Prockop, Intravenous hMSCs improve myocardial infarction in mice because cells embolized in lung are activated to secrete the anti-inflammatory protein TSG-6. Cell Stem Cell, 2009. 5(1): p. 54-63.
29. Polchert, D., J. Sobinsky, G. Douglas, M. Kidd, A. Moadsiri, E. Reina, K. Genrich, S. Mehrotra, S. Setty, B. Smith, and A. Bartholomew, IFN-gamma activation of mesenchymal stem cells for treatment and prevention of graft versus host disease. Eur. J. Immunol., 2008. 38(6): p. 1745-55.
30. Ren, G., X. Chen, F. Dong, W. Li, X. Ren, Y. Zhang, and Y. Shi, Concise review: mesenchymal stem cells and translational medicine: emerging issues. Stem Cells Transl Med, 2012. 1(1): p. 51-8.
31. Zappia, E., S. Casazza, E. Pedemonte, F. Benvenuto, I. Bonanni, E. Gerdoni, D. Giunti, A. Ceravolo, F. Cazzanti, F. Frassoni, G. Mancardi, and A. Uccelli, Mesenchymal stem cells ameliorate experimental autoimmune encephalomyelitis inducing T-cell anergy. Blood, 2005. 106(5): p. 1755-61.
32. Aggarwal, S. and M.F. Pittenger, Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood, 2005. 105(4): p. 1815-22.
33. Mohammadzadeh, A., A.A. Pourfathollah, S. Shahrokhi, S.M. Hashemi, S.L. Moradi, and M. Soleimani, Immunomodulatory effects of adipose-derived mesenchymal stem cells on the gene expression of major transcription factors of T cell subsets. Int. Immunopharmacol., 2014. 20(2): p. 316-21.
34. Qi, K., N. Li, Z. Zhang, and G. Melino, Tissue regeneration: The crosstalk between mesenchymal stem cells and immune response. Cell. Immunol., 2018. 326: p. 86-93.
35. Frenette, P.S., S. Pinho, D. Lucas, and C. Scheiermann, Mesenchymal stem cell: keystone of the hematopoietic stem cell niche and a stepping-stone for regenerative medicine. Annu. Rev. Immunol., 2013. 31: p. 285-316.
36. Ren, G., L. Zhang, X. Zhao, G. Xu, Y. Zhang, A.I. Roberts, R.C. Zhao, and Y. Shi, Mesenchymal stem cell-mediated immunosuppression occurs via concerted action of chemokines and nitric oxide. Cell Stem Cell, 2008. 2(2): p. 141-50.
37. Li, W., G. Ren, Y. Huang, J. Su, Y. Han, J. Li, X. Chen, K. Cao, Q. Chen, P. Shou, L. Zhang, Z.R. Yuan, A.I. Roberts, S. Shi, A.D. Le, and Y. Shi, Mesenchymal stem cells: a double-edged sword in regulating immune responses. Cell Death Differ., 2012. 19(9): p. 1505-13.
38. Takeda, K., T.L. Webb, F. Ning, Y. Shiraishi, D.P. Regan, L. Chow, M.J. Smith, S. Ashino, A.M. Guth, S. Hopkins, E.W. Gelfand, and S. Dow, Mesenchymal Stem Cells Recruit CCR2(+) Monocytes To Suppress Allergic Airway Inflammation. J. Immunol., 2018. 200(4): p. 1261-1269.
39. Cho, K.S., M.K. Park, S.A. Kang, H.Y. Park, S.L. Hong, H.K. Park, H.S. Yu, and H.J. Roh, Adipose-derived stem cells ameliorate allergic airway inflammation by inducing regulatory T cells in a mouse model of asthma. Mediators Inflamm., 2014. 2014: p. 436476.
40. Bonfield, T.L., M. Koloze, D.P. Lennon, B. Zuchowski, S.E. Yang, and A.I. Caplan, Human mesenchymal stem cells suppress chronic airway inflammation in the murine ovalbumin asthma model. Am. J. Physiol. Lung Cell Mol. Physiol., 2010. 299(6): p. L760-70.
41. Kavanagh, H. and B.P. Mahon, Allogeneic mesenchymal stem cells prevent allergic airway inflammation by inducing murine regulatory T cells. Allergy, 2011. 66(4): p. 523-31.
42. Davies, L.C., N. Heldring, N. Kadri, and K. Le Blanc, Mesenchymal Stromal Cell Secretion of Programmed Death-1 Ligands Regulates T Cell Mediated Immunosuppression. Stem Cells, 2017. 35(3): p. 766-776.
43. Yoo, H.S., K. Lee, K. Na, Y.X. Zhang, H.J. Lim, T. Yi, S.U. Song, and M.S. Jeon, Mesenchymal stromal cells inhibit CD25 expression via the mTOR pathway to potentiate T-cell suppression. Cell Death Dis., 2017. 8(2): p. e2632.
44. Chan, C.K., T.C. Lin, Y.A. Huang, Y.S. Chen, C.L. Wu, H.Y. Lo, M.L. Kuo, K.H. Wu, and J.L. Huang, The modulation of Th2 immune pathway in the immunosuppressive effect of human umbilical cord mesenchymal stem cells in a murine asthmatic model. Inflamm. Res., 2016. 65(10): p. 795-801.
45. Secunda, R., R. Vennila, A.M. Mohanashankar, M. Rajasundari, S. Jeswanth, and R. Surendran, Isolation, expansion and characterisation of mesenchymal stem cells from human bone marrow, adipose tissue, umbilical cord blood and matrix: a comparative study. Cytotechnology, 2015. 67(5): p. 793-807.
46. English, K., Mechanisms of mesenchymal stromal cell immunomodulation. Immunol. Cell Biol., 2013. 91(1): p. 19-26.
47. Madec, A.M., R. Mallone, G. Afonso, E. Abou Mrad, A. Mesnier, A. Eljaafari, and C. Thivolet, Mesenchymal stem cells protect NOD mice from diabetes by inducing regulatory T cells. Diabetologia, 2009. 52(7): p. 1391-9.
48. Augello, A., R. Tasso, S.M. Negrini, R. Cancedda, and G. Pennesi, Cell therapy using allogeneic bone marrow mesenchymal stem cells prevents tissue damage in collagen-induced arthritis. Arthritis Rheum., 2007. 56(4): p. 1175-86.
49. Klinker, M.W. and C.H. Wei, Mesenchymal stem cells in the treatment of inflammatory and autoimmune diseases in experimental animal models. World J. Stem Cells, 2015. 7(3): p. 556-67.
50. Ferrara, J.L., J.E. Levine, P. Reddy, and E. Holler, Graft-versus-host disease. Lancet, 2009. 373(9674): p. 1550-61.
51. Amorin, B., A.P. Alegretti, V. Valim, A. Pezzi, A.M. Laureano, M.A. da Silva, A. Wieck, and L. Silla, Mesenchymal stem cell therapy and acute graft-versus-host disease: a review. Hum. Cell, 2014. 27(4): p. 137-50.
52. Goodwin, M., V. Sueblinvong, P. Eisenhauer, N.P. Ziats, L. LeClair, M.E. Poynter, C. Steele, M. Rincon, and D.J. Weiss, Bone marrow-derived mesenchymal stromal cells inhibit Th2-mediated allergic airways inflammation in mice. Stem Cells, 2011. 29(7): p. 1137-48.
53. Sagaradze, G., O. Grigorieva, P. Nimiritsky, N. Basalova, N. Kalinina, Z. Akopyan, and A. Efimenko, Conditioned Medium from Human Mesenchymal Stromal Cells: Towards the Clinical Translation. Int. J. Mol. Sci., 2019. 20(7).
54. Kay, A.G., G. Long, G. Tyler, A. Stefan, S.J. Broadfoot, A.M. Piccinini, J. Middleton, and O. Kehoe, Mesenchymal Stem Cell-Conditioned Medium Reduces Disease Severity and Immune Responses in Inflammatory Arthritis. Sci. Rep., 2017. 7(1): p. 18019.
55. Chen, Z.Y., Y.Y. Hu, X.F. Hu, and L.X. Cheng, The conditioned medium of human mesenchymal stromal cells reduces irradiation-induced damage in cardiac fibroblast cells. J. Radiat. Res., 2018. 59(5): p. 555-564.
56. Seetharaman, R., A. Mahmood, P. Kshatriya, D. Patel, and A. Srivastava, Mesenchymal Stem Cell Conditioned Media Ameliorate Psoriasis Vulgaris: A Case Study. Case Rep Dermatol Med, 2019. 2019: p. 8309103.
57. Zeng, S.L., L.H. Wang, P. Li, W. Wang, and J. Yang, Mesenchymal stem cells abrogate experimental asthma by altering dendritic cell function. Mol Med Rep, 2015. 12(2): p. 2511-20.
58. Braza, F., S. Dirou, V. Forest, V. Sauzeau, D. Hassoun, J. Chesne, M.A. Cheminant-Muller, C. Sagan, A. Magnan, and P. Lemarchand, Mesenchymal Stem Cells Induce Suppressive Macrophages Through Phagocytosis in a Mouse Model of Asthma. Stem Cells, 2016. 34(7): p. 1836-45.
59. Bai, L., D.P. Lennon, V. Eaton, K. Maier, A.I. Caplan, S.D. Miller, and R.H. Miller, Human bone marrow-derived mesenchymal stem cells induce Th2-polarized immune response and promote endogenous repair in animal models of multiple sclerosis. Glia, 2009. 57(11): p. 1192-203.
60. Duffy, M.M., T. Ritter, R. Ceredig, and M.D. Griffin, Mesenchymal stem cell effects on T-cell effector pathways. Stem Cell. Res. Ther., 2011. 2(4): p. 34.
61. Zhou, Y., A. Day, S. Haykal, A. Keating, and T.K. Waddell, Mesenchymal stromal cells augment CD4+ and CD8+ T-cell proliferation through a CCL2 pathway. Cytotherapy, 2013. 15(10): p. 1195-207.
62. Rasmusson, I., O. Ringden, B. Sundberg, and K. Le Blanc, Mesenchymal stem cells inhibit lymphocyte proliferation by mitogens and alloantigens by different mechanisms. Exp. Cell Res., 2005. 305(1): p. 33-41.
63. Sato, K., K. Ozaki, I. Oh, A. Meguro, K. Hatanaka, T. Nagai, K. Muroi, and K. Ozawa, Nitric oxide plays a critical role in suppression of T-cell proliferation by mesenchymal stem cells. Blood, 2007. 109(1): p. 228-34.
64. Su, J., X. Chen, Y. Huang, W. Li, J. Li, K. Cao, G. Cao, L. Zhang, F. Li, A.I. Roberts, H. Kang, P. Yu, G. Ren, W. Ji, Y. Wang, and Y. Shi, Phylogenetic distinction of iNOS and IDO function in mesenchymal stem cell-mediated immunosuppression in mammalian species. Cell Death Differ., 2014. 21(3): p. 388-96.
65. Balkhi, M.Y., Q. Ma, S. Ahmad, and R.P. Junghans, T cell exhaustion and Interleukin 2 downregulation. Cytokine, 2015. 71(2): p. 339-47.