系統識別號 U0026-1908201517012200
論文名稱(中文) 單核球細胞與巨噬細胞之間的TLR7/8活化的差異
論文名稱(英文) The differences in TLR7/8 activation between monocytes and macrophages
校院名稱 成功大學
系所名稱(中) 醫學檢驗生物技術學系
系所名稱(英) Department of Medical Laboratory Science and Biotechnology
學年度 103
學期 2
出版年 104
研究生(中文) 許苑瑩
研究生(英文) Yuan-Ying Hsu
學號 T36021099
學位類別 碩士
語文別 英文
論文頁數 96頁
口試委員 指導教授-林尊湄
中文關鍵字 TLR7  TLR8  單核球  巨噬細胞  ISG  NF-κB  THP-1  PMA 
英文關鍵字 TLR7  TLR8  monocyte  macrophage  ISG  NF-κB  THP-1  PMA 
中文摘要 在先天性免疫系統中,單核球和巨噬細胞是主要的吞噬細胞,藉由表現在endosome胞器的Toll-like receptors (TLRs),可以辨識吞噬之病原體的核酸,進而啟動免疫防禦功能。當活化TLRs時,會導致吞噬細胞的訊息傳遞,促使一些NF-κB相關的發炎性激素和第一型干擾素基因的表現。因TLR7和TLR8的基因都位於X染色體,兩者在系統發育是相當相似的,且都具有辨識單股RNA的能力;曾經有研究證明,在TLRs轉染到HEK293細胞後,TLR8會抑制TLR7活性,但是TLR7不會抑制TLR8的作用,並且如果小老鼠缺乏TLR8就會導致自體免疫疾病的發生;然而至今,在單核球和巨噬細胞中,在同源性刺激物的刺激下,TLR7和TLR8之間的調控作用與發炎反應產生的機制目前還不是很清楚。因此,本研究的主要目的是探討單核球細胞與巨噬細胞之間的TLR7和TLR8活化的差異,並釐清兩種細胞中TLR7和TLR8功能差異。由我們的結果發現,單核球中R848 (TLR7/8刺激物)和CL075 (TLR8/7刺激物)具有活化NF-κB和interferon regulatory factor (IRF)訊息之能力,且隨著劑量而上升;但是對於TLR7刺激劑(R837和Loxoribone)卻沒有活化的能力。因此,在THP-1細胞中,我們證實若抑制TLR8表現或增加TLR7表現時,單核球細胞對TLR7誘發的NF-κB活性可以分別增加1.4±0.144倍和4.5±1.05倍。此外,我們也證實在巨噬細胞中,TLR7和TLR8刺激物都可以誘發NF-κB活化,但是卻沒有辦法活化interferon-stimulated gene (ISG)反應;進一步實驗證實,在巨噬細胞中MyD88、IRAK1、IRAK4和IRF7的表現明顯比單核球減少,但是TLR7和TLR8 mRNA的表現卻分別增加3.14±0.42倍和3.97±1.19倍。同時,我們發現利用PMA刺激單核球分化為巨噬細胞的過程中,會誘發PKC-NF-κB-STAT3訊息路徑的活化,導致NF-κB和ISG的基本活性會分別增加1.79±0.49倍和4.44±1.54倍,但巨噬細胞在Loxoribine、R848和CL075刺激之下,只有發炎性細胞激素TNF-α、IL-6、IL-12p40 mRNA的表現大幅度地被增加,而MyD88 和IRF7 mRNA的表現卻是顯著地被減少。因此,我們認為TLR7和TLR8在單核球和巨噬細胞中調控著不同的免疫反應,巨噬細胞顯然比單核球傾向發炎反應誘發的能力。總而言之,我們的研究證明,在單核球中TLR7和TLR8之間存在交互作用和調控關係,以維持吞噬細胞的免疫平衡;在巨噬細胞中,TLR7和TLR8誘導ISG訊息路徑的完全受損,因此導致巨噬細胞比較傾向發炎反應的進行,這或許就是為何慢性感染病患會產生較嚴重的發炎反應的原因。
英文摘要 Monocytes and macrophages are essential phagocytes of the innate immune system. The Toll-like receptors (TLRs) of endosome are important in sensing foreign nucleic acids encountered by phagocytes. TLRs activation leads to the production of a variety of nuclear factor (NF)-κB-mediated cytokines and type I interferons against pathogens. TLR7 and TLR8, phylogenetically similar genes located in X-chromosome, are both capable of recognizing single-stranded RNA. Previous study indicated that TLR8 inhibited TLR7 but not vice versa in HEK293 cells transfected with TLRs, and TLR8 deficiency led to the development of autoimmune disease in mice. However, the role of TLR7 and TLR8 in modulating the immune response for their cognate agonists in monocytes and macrophages is still not clear. The aim of this study is to investigate the regulation and interaction between TLR7 and TLR8 in order to clarify their differences between monocytes and macrophages. Our data indicated that R848 (TLR7/8 agonist) and CL075 (TLR8/7 agonist) could activate both NF-κB and interferon regulatory factor (IRF)-mediated pathways dose-dependently, but failed to respond for TLR7 agonists (R837 and Loxoribine) in THP-1 cells. We demonstrated the silent TLR8 and enhanced TLR7 expression could increase TLR7-induced NF-κB activation in monocytes by 1.4±0.144 and 4.5±1.05 folds, respectively. In addition, the TLR7 and TLR8 agonists could induce NF-κB activation but no interferon-stimulated gene (ISG) response in macrophages. However, TLR7 and TLR8 activation signalings were impaired with significantly reduced levels of MyD88, IRAK1, IRAK4, and IRF7, but the levels of TLR7 and TLR8 mRNA were increased by 3.14±0.42 and 3.97±1.19 folds in macrophages as compared with those of monocytes. The PKC-NF-κB-STAT3 pathway was involved in basal NF-κB and ISG activation to elevate 1.79±0.49 and 4.44±1.54 folds when monocytes were stimulated to differentiate into macrophages by PMA. Furthermore, mRNA levels of pro-inflammatory cytokine, TNF-α, IL-6, and IL-12p40 were dramatically elevated, whereas MyD88 and IRF7 were significantly reduced in macrophages upon Loxoribine, R848 and CL075 stimulation. Therefore, TLR7 and TLR8 might modulate different immune responses in monocytes and macrophages, and macrophages preferred to induce inflammatory response. In conclusion, our findings provide evidence that the interaction between TLR7 and TLR8 is regulated to maintain immune balance in monocytes. TLR7- and TLR8-induced ISG signalings are impaired and NF-κB-induced inflammation is increased in macrophages. Thus, it can explain why severe inflammatory responses occurred in patients with chronic infection.
論文目次 Abstract (in Chinese)......................I
Abstract (in English)................... III
Table List...............................VII
Figure List.............................VIII
Appendix List..............................X
Materials and Methods...................8-38
Discussion and Conclusions.............45-50
參考文獻 1. Akira S, Hemmi H. Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett 2003,85:85-95.
2. Kawai T, Akira S. TLR signaling. Cell Death Differ 2006,13:816-825.
3. Medzhitov R, Janeway CA, Jr. Innate immunity: impact on the adaptive immune response. Curr Opin Immunol 1997,9:4-9.
4. Iwasaki A, Medzhitov R. Toll-like receptor control of the adaptive immune responses. Nat Immunol 2004,5:987-995.
5. Kaisho T, Akira S. Toll-like receptors and their signaling mechanism in innate immunity. Acta Odontol Scand 2001,59:124-130.
6. Zhu J, Mohan C. Toll-like receptor signaling pathways--therapeutic opportunities. Mediators Inflamm 2010,2010:781235.
7. Kawai T, Akira S. TLR signaling. Semin Immunol 2007,19:24-32.
8. Slack JL, Schooley K, Bonnert TP, Mitcham JL, Qwarnstrom EE, Sims JE, et al. Identification of two major sites in the type I interleukin-1 receptor cytoplasmic region responsible for coupling to pro-inflammatory signaling pathways. J Biol Chem 2000,275:4670-4678.
9. Parihar A, Eubank TD, Doseff AI. Monocytes and macrophages regulate immunity through dynamic networks of survival and cell death. J Innate Immun 2010,2:204-215.
10. Auffray C, Sieweke MH, Geissmann F. Blood monocytes: development, heterogeneity, and relationship with dendritic cells. Annu Rev Immunol 2009,27:669-692.
11. Fahy RJ, Doseff AI, Wewers MD. Spontaneous human monocyte apoptosis utilizes a caspase-3-dependent pathway that is blocked by endotoxin and is independent of caspase-1. J Immunol 1999,163:1755-1762.
12. Bender AT, Ostenson CL, Giordano D, Beavo JA. Differentiation of human monocytes in vitro with granulocyte-macrophage colony-stimulating factor and macrophage colony-stimulating factor produces distinct changes in cGMP phosphodiesterase expression. Cell Signal 2004,16:365-374.
13. Tosi MF. Innate immune responses to infection. J Allergy Clin Immunol 2005,116:241-249; quiz 250.
14. Gordon S. Alternative activation of macrophages. Nat Rev Immunol 2003,3:23-35.
15. Schmitz F, Mages J, Heit A, Lang R, Wagner H. Transcriptional activation induced in macrophages by Toll-like receptor (TLR) ligands: from expression profiling to a model of TLR signaling. Eur J Immunol 2004,34:2863-2873.
16. Linker R, Gold R, Luhder F. Function of neurotrophic factors beyond the nervous system: inflammation and autoimmune demyelination. Crit Rev Immunol 2009,29:43-68.
17. Hoffmann JA. The immune response of Drosophila. Nature 2003,426:33-38.
18. Lemaitre B, Nicolas E, Michaut L, Reichhart JM, Hoffmann JA. The dorsoventral regulatory gene cassette spatzle/Toll/cactus controls the potent antifungal response in Drosophila adults. Cell 1996,86:973-983.
19. Medzhitov R, Preston-Hurlburt P, Janeway CA, Jr. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature 1997,388:394-397.
20. Krishnan J, Selvarajoo K, Tsuchiya M, Lee G, Choi S. Toll-like receptor signal transduction. Exp Mol Med 2007,39:421-438.
21. O'Neill LA, Golenbock D, Bowie AG. The history of Toll-like receptors - redefining innate immunity. Nat Rev Immunol 2013,13:453-460.
22. Saitoh S, Miyake K. Regulatory molecules required for nucleotide-sensing Toll-like receptors. Immunol Rev 2009,227:32-43.
23. Nishiya T, DeFranco AL. Ligand-regulated chimeric receptor approach reveals distinctive subcellular localization and signaling properties of the Toll-like receptors. J Biol Chem 2004,279:19008-19017.
24. Takeuchi O, Kawai T, Muhlradt PF, Morr M, Radolf JD, Zychlinsky A, et al. Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int Immunol 2001,13:933-940.
25. Alexopoulou L, Thomas V, Schnare M, Lobet Y, Anguita J, Schoen RT, et al. Hyporesponsiveness to vaccination with Borrelia burgdorferi OspA in humans and in TLR1- and TLR2-deficient mice. Nat Med 2002,8:878-884.
26. Alexopoulou L, Holt AC, Medzhitov R, Flavell RA. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 2001,413:732-738.
27. Hoshino K, Takeuchi O, Kawai T, Sanjo H, Ogawa T, Takeda Y, et al. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J Immunol 1999,162:3749-3752.
28. Shimazu R, Akashi S, Ogata H, Nagai Y, Fukudome K, Miyake K, et al. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med 1999,189:1777-1782.
29. Hayashi F, Smith KD, Ozinsky A, Hawn TR, Yi EC, Goodlett DR, et al. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 2001,410:1099-1103.
30. Diebold SS, Kaisho T, Hemmi H, Akira S, Reis e Sousa C. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 2004,303:1529-1531.
31. Heil F, Hemmi H, Hochrein H, Ampenberger F, Kirschning C, Akira S, et al. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science 2004,303:1526-1529.
32. Lund JM, Alexopoulou L, Sato A, Karow M, Adams NC, Gale NW, et al. Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc Natl Acad Sci U S A 2004,101:5598-5603.
33. Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, et al. A Toll-like receptor recognizes bacterial DNA. Nature 2000,408:740-745.
34. Ohto U, Shibata T, Tanji H, Ishida H, Krayukhina E, Uchiyama S, et al. Structural basis of CpG and inhibitory DNA recognition by Toll-like receptor 9. Nature 2015,520:702-705.
35. Takeda K, Akira S. TLR signaling pathways. Semin Immunol 2004,16:3-9.
36. Schoenemeyer A, Barnes BJ, Mancl ME, Latz E, Goutagny N, Pitha PM, et al. The interferon regulatory factor, IRF5, is a central mediator of toll-like receptor 7 signaling. J Biol Chem 2005,280:17005-17012.
37. Bergstrom B, Aune MH, Awuh JA, Kojen JF, Blix KJ, Ryan L, et al. TLR8 Senses Staphylococcus aureus RNA in Human Primary Monocytes and Macrophages and Induces IFN-beta Production via a TAK1-IKKbeta-IRF5 Signaling Pathway. 2015,195:1100-1111.
38. Doyle S, Vaidya S, O'Connell R, Dadgostar H, Dempsey P, Wu T, et al. IRF3 mediates a TLR3/TLR4-specific antiviral gene program. Immunity 2002,17:251-263.
39. Du X, Poltorak A, Wei Y, Beutler B. Three novel mammalian toll-like receptors: gene structure, expression, and evolution. Eur Cytokine Netw 2000,11:362-371.
40. Forsbach A, Nemorin JG, Montino C, Muller C, Samulowitz U, Vicari AP, et al. Identification of RNA sequence motifs stimulating sequence-specific TLR8-dependent immune responses. J Immunol 2008,180:3729-3738.
41. Krieg AM. The toll of too much TLR7. Immunity 2007,27:695-697.
42. Jurk M, Heil F, Vollmer J, Schetter C, Krieg AM, Wagner H, et al. Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat Immunol 2002,3:499.
43. Wang J, Shao Y, Bennett TA, Shankar RA, Wightman PD, Reddy LG. The functional effects of physical interactions among Toll-like receptors 7, 8, and 9. J Biol Chem 2006,281:37427-37434.
44. Desnues B, Macedo AB, Roussel-Queval A, Bonnardel J, Henri S, Demaria O, et al. TLR8 on dendritic cells and TLR9 on B cells restrain TLR7-mediated spontaneous autoimmunity in C57BL/6 mice. Proc Natl Acad Sci U S A 2014,111:1497-1502.
45. Kurihara Y, Nakahara T, Furue M. alphaVbeta3-integrin expression through ERK activation mediates cell attachment and is necessary for production of tumor necrosis factor alpha in monocytic THP-1 cells stimulated by phorbol myristate acetate. Cell Immunol 2011,270:25-31.
46. Zhang C, Bai N, Chang A, Zhang Z, Yin J, Shen W, et al. ATF4 is directly recruited by TLR4 signaling and positively regulates TLR4-trigged cytokine production in human monocytes. Cell Mol Immunol 2013,10:84-94.
47. Oh DR, Kang HW, Kim JR, Kim S, Park IK, Rhee JH, et al. PMA induces vaccine adjuvant activity by the modulation of TLR signaling pathway. Mediators Inflamm 2014,2014:406514.
48. Jones BW, Means TK, Heldwein KA, Keen MA, Hill PJ, Belisle JT, et al. Different Toll-like receptor agonists induce distinct macrophage responses. J Leukoc Biol 2001,69:1036-1044.
49. Kogut MH, Genovese KJ, He H, Kaiser P. Flagellin and lipopolysaccharide up-regulation of IL-6 and CXCLi2 gene expression in chicken heterophils is mediated by ERK1/2-dependent activation of AP-1 and NF-kappaB signaling pathways. Innate Immun 2008,14:213-222.
50. Demaria O, Pagni PP, Traub S, de Gassart A, Branzk N, Murphy AJ, et al. TLR8 deficiency leads to autoimmunity in mice. J Clin Invest 2010,120:3651-3662.
51. Guiducci C, Gong M, Cepika AM, Xu Z, Tripodo C, Bennett L, et al. RNA recognition by human TLR8 can lead to autoimmune inflammation. J Exp Med 2013,210:2903-2919.
52. Spitzer JH, Visintin A, Mazzoni A, Kennedy MN, Segal DM. Toll-like receptor 1 inhibits Toll-like receptor 4 signaling in endothelial cells. Eur J Immunol 2002,32:1182-1187.
53. Kurihara Y, Furue M. Interferon-gamma enhances phorbol myristate acetate-induced cell attachment and tumor necrosis factor production via the NF-kappaB pathway in THP-1 human monocytic cells. Mol Med Rep 2013,7:1739-1744.
54. Revie D, Salahuddin SZ. Role of macrophages and monocytes in hepatitis C virus infections. World J Gastroenterol 2014,20:2777-2784.
55. Dolganiuc A, Garcia C, Kodys K, Szabo G. Distinct Toll-like receptor expression in monocytes and T cells in chronic HCV infection. World J Gastroenterol 2006,12:1198-1204.
56. Wynn TA, Chawla A, Pollard JW. Macrophage biology in development, homeostasis and disease. Nature 2013,496:445-455.
57. Stoger JL, Goossens P, de Winther MP. Macrophage heterogeneity: relevance and functional implications in atherosclerosis. Curr Vasc Pharmacol 2010,8:233-248.
58. Lee KM, Yin C, Verschoor CP, Bowdish DME. Macrophage Function Disorders. In: eLS: John Wiley & Sons, Ltd; 2001.
59. Kang JW, Park YS, Lee DH, Kim JH, Kim MS, Bak Y, et al. Intracellular interaction of interleukin (IL)-32alpha with protein kinase Cepsilon (PKCepsilon ) and STAT3 protein augments IL-6 production in THP-1 promonocytic cells. J Biol Chem 2012,287:35556-35564.
60. Giraud S, Bienvenu F, Avril S, Gascan H, Heery DM, Coqueret O. Functional interaction of STAT3 transcription factor with the coactivator NcoA/SRC1a. J Biol Chem 2002,277:8004-8011.
61. Abell K, Watson CJ. The Jak/Stat pathway: a novel way to regulate PI3K activity. Cell Cycle 2005,4:897-900.
62. Yu X, Kennedy RH, Liu SJ. JAK2/STAT3, not ERK1/2, mediates interleukin-6-induced activation of inducible nitric-oxide synthase and decrease in contractility of adult ventricular myocytes. J Biol Chem 2003,278:16304-16309.
63. Du Z, Shen Y, Yang W, Mecklenbrauker I, Neel BG, Ivashkiv LB. Inhibition of IFN-alpha signaling by a PKC- and protein tyrosine phosphatase SHP-2-dependent pathway. Proc Natl Acad Sci U S A 2005,102:10267-10272.
64. Graff JW, Dickson AM, Clay G, McCaffrey AP, Wilson ME. Identifying functional microRNAs in macrophages with polarized phenotypes. J Biol Chem 2012,287:21816-21825.
65. Liu G, Abraham E. MicroRNAs in immune response and macrophage polarization. Arterioscler Thromb Vasc Biol 2013,33:170-177.
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