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系統識別號 U0026-2808201708370700
論文名稱(中文) 血鐵蛋白與細胞激素生成在登革病毒感染中所扮演的角色
論文名稱(英文) The role of ferritin and cytokine production in dengue infection
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 105
學期 2
出版年 106
研究生(中文) 陳又菱
研究生(英文) Yu-Ling Chen
學號 S46044111
學位類別 碩士
語文別 英文
論文頁數 69頁
口試委員 指導教授-劉清泉
口試委員-劉校生
口試委員-張志鵬
口試委員-李英瑞
中文關鍵字 登革熱病毒  血鐵蛋白  介白素-6  腫瘤壞死因子-α  介白素-10  由U937所衍生的巨噬細胞  CD71  CD365  細胞自噬 
英文關鍵字 Dengue virus  ferritin  IL-6  TNF-α  IL-10  U937-derived macrophages  CD71  CD365  autophagy 
學科別分類
中文摘要 登革熱病毒是屬於黃熱病毒科的病毒,為單股正股的核糖核酸病毒,透過蚊媒的傳播病原體造成感染和在全球的擴散。受到登革熱病毒感染所造成的廣泛臨床症狀,有輕微發燒到致死的出血性疾病。根據先前研究指出,在人體血清中高濃度的血鐵蛋白,與登革病毒的疾病嚴重程度有關聯,在嚴重登革病患的血液中,血鐵蛋白濃度會顯著增高。而血鐵蛋白為人體中廣為人知之的鐵儲存蛋白,可由大多數細胞製造,在人體的血液中,巨噬細胞為血鐵蛋白的主要製造者之一。另外,血清中的血鐵蛋白為巨噬細胞活化因子之一。在本研究中,我們目標在釐清於嚴重的登革病患中,血鐵蛋白所扮演的角色。我們發現在2015年台南登革大流行中,登革病患的血液中血鐵蛋白濃度明顯的增加。此外,血液中的血鐵蛋白的濃度與臨床疾病的嚴重性有正相關性。為了瞭解在登革病毒感染下,血鐵蛋白的角色和機制,我們首先建立了由PMA刺激下形成的U937衍生的巨噬細胞模式。我們確定登革病毒蛋白: capsid、NS1及NS3會在細胞實驗中表現。額外給予U937衍生的巨噬細胞血鐵重組蛋白、血鐵重組重鏈蛋白及血鐵重組輕鏈蛋白後,介白素-6、腫瘤壞死因子-α、介白素-10的分泌會增加。使用shRNA阻斷血鐵重鍊蛋白及血鐵輕鍊蛋白表現,會抑制細胞激素產生。血鐵蛋白會透過CD71與CD365增加細胞激素產生。給予3-MA會降低血鐵蛋白、介白素-6、腫瘤壞死因子-α及介白素-10分泌。我們亦証實在登革熱病毒感染引發的細胞自噬與血鐵蛋白的產生有關連性。總結來說,在細胞實驗中,血鐵蛋白在調控介白素-6、腫瘤壞死因子-α、介白素-10表現,在登革病毒感染的致病機轉中扮演重要的角色。
英文摘要 Dengue virus (DENV), a single positive-stranded RNA virus of the family Flaviviridae, is an emerging mosquito-borne infection worldwide. Dengue infection results in broad clinical symptoms ranging from mild fever to fatal hemorrhagic disease. According to previous findings, high serum ferritin levels are correlated with severity of dengue infection. The circulating ferritin is significantly elevated in severe dengue patients. Ferritin is a well-known iron-storage protein produced by most cells in the body. In the human blood, activated macrophages are major producers of ferritin. Thus, ferritin is one of the macrophage activation markers. In this study, we aimed to evaluate the roles of ferritin in the pathogenesis of severe dengue. Using clinical samples collected from patients in 2015 Tainan dengue outbreak, we found that plasma ferritin levels were elevated in dengue patients. Furthermore, the plasma ferritin elevation accompanied with disease severity. To investigate the roles and the mechanisms of ferritin in DENV infection, we first established a propylene glycol methyl ether acetate (PMA)-induced U937-derived macrophage model. Capsid, NS1 and NS3 protein expression were confirmed in vitro model. Treatments of ferritin, ferritin heavy chain (FTH) and ferritin light chain (FTL) enhanced IL-6, TNF-α, and IL-10.The shRNA knockdown of FTH and FTL inhibited cytokine expressions. Furthermore, ferritin increased cytokine via CD71 and CD365. Treating with 3-MA (autophagy inhibitor) decreased ferritin, IL-6, TNF-α, and IL-10. Ferritin production may relate to autophagy in DENV infection. In conclusion, ferritin regulates the levels of IL-6, TNF-α, and IL-10and may play role in the pathogenesis of dengue infection.

論文目次 Table of Contents
Abstract in Chinese I
Abstract II
Acknowledgement III
Figure List VII
Abbreviations IX
Introduction 1
Objective and Specific Aims 13
1. To clarify whether U937-derived macrophages are infected by DENV. 13
2. To confirm whether ferritin and cytokines are secreted from DENV-infected U937-derived macrophages. 14
3. To investigate whether cytokine production can be regulated by ferritin. 14
4. To investigate the effect of CD71 and CD365 neutralizing antibodies on cytokine production after DENV infection. 14
5. To check whether the secretion of ferritin is through autophagy. 14
A. Materials 15
A-1 Cell lines 15
A-2 Viruses 15
A-3 Chemicals and reagents 15
A-4 Antibodies 18
A-5 Kits 19
A-6 Recombinant Proteins 19
A-7 Experimental consumables 20
A-8 Instruments 21
A-9 shRNA plasmid 22
B. Methods 24
B-1 Cell cultures 24
B-2 Virus culture 24
B-3 Plaque assay 25
B-4 UV-inactivated DENV 25
B-5 Western blotting 25
B-6 Lentiviral-based RNAi gene knockdown 26
B-7 Monocyte activation 27
B-8 Flow cytometry analysis for monocytes activation 27
B-9 Confocal assay 27
B-10 DENV infection 28
B-11 Detection of cytokines in cultured supernatants 28
B-12 Statistics 29
Results 30
1. DENV infected U937-derived macrophage models. 30
1.1 Set up a U937-derived macrophage model. 30
1.2 Set up a DENV infected U937-derived macrophage model.. 30
2. DENV infection induced the production of ferritin and cytokines. 31
2.1 Investigation of whether ferritin is secreted from DENV-infected U937-derived macrophages. 31
2.2 The increase or decrease of ferritin required the replication of DENV. 31
2.3 The cytokine productions in DENV-infected U937-derived macrophages. 32
3. Ferritin was responsible for cytokine production. 32
3.1 The role of ferritin heavy chain (FTH) on cytokine production.. 32
3.2 The role of ferritin light chain (FTL) on cytokine production.. 33
3.3 Ferritin, FTH, and FTL could dose-dependently and time-dependently induce cytokine production.. 34
4. Two candidate receptors may mediate cytokine production by ferritin stimulation.. 35
4.1 Ferritin induced cytokine production through CD71 in U937-derived macrophages.. 35
4.2 Ferritin induced cytokine production through CD365 in U937-derived macrophages. 35
4.3 The combination of anti-CD71 antibody and anti-CD365 antibody treated on U937-derived macrophages. 36
5. Ferritin was associated with DENV-induced autophagy and cytokine production. 37
5.1 The ferritin was regulated by autophagy-mediated secretory pathway under DENV infection. 37
5.2 DENV-induced autophagy influenced cytokine secretion. 38
Discussion 39
Conclusions 43
References 44
Figures and Legends 49
Appendix 69
Curriculum Vitae70


參考文獻 Ab-Rahman, H. A., Rahim, H., AbuBakar, S., & Wong, P.-F. (2016). Macrophage activation syndrome-associated markers in severe dengue. International Journal of Medical Sciences, 13, 179.
Ackermann, M., & Padmanabhan, R. (2001). De novo synthesis of RNA by the dengue virus RNA-dependent RNA polymerase exhibits temperature dependence at the initiation but not elongation phase. Journal of Biological Chemistry, 276, 39926-39937.
Arshad, U., Ahmad, S. Q., & Khan, F. (2015). Hemophagocytic lymphohistiocytosis in a patient with dengue infection. Hematology/oncology and stem cell therapy, 8, 189-190.
Bhatt, S., Gething, P. W., Brady, O. J., Messina, J. P., Farlow, A. W., Moyes, C. L., & Sankoh, O. (2013). The global distribution and burden of dengue. Nature, 496, 504-507.
Billiau, A. D., Roskams, T., Van Damme-Lombaerts, R., Matthys, P., & Wouters, C. (2005). Macrophage activation syndrome: characteristic findings on liver biopsy illustrating the key role of activated, IFN-γ-producing lymphocytes and IL-6-and TNF-α-producing macrophages. Blood, 105, 1648-1651.
Carswell, E., Old, L. J., Kassel, R., Green, S., Fiore, N., & Williamson, B. (1975). An endotoxin-induced serum factor that causes necrosis of tumors. Proceedings of the National Academy of Sciences, 72, 3666-3670.
Chang, S.-C. (2015). Raising clinical awareness for better dengue fever outbreak control. Journal of the Formosan Medical Association, 114, 1025-1026.
Chaturvedi, U., Agarwal, R., Elbishbishi, E., & Mustafa, A. (2000). Cytokine cascade in dengue hemorrhagic fever: implications for pathogenesis. Pathogens and Disease, 28, 183-188.
Chen, L.-C., Lei, H.-Y., Liu, C.-C., Shiesh, S.-C., Chen, S.-H., Liu, H.-S., & Yeh, T.-M. (2006). Correlation of serum levels of macrophage migration inhibitory factor with disease severity and clinical outcome in dengue patients. The American Journal of Tropical Medicine and Hygiene, 74, 142-147.
Chen, T. T., Li, L., Chung, D.-H., Allen, C. D., Torti, S. V., Torti, F. M., & Niemi, E. C. (2005). TIM-2 is expressed on B cells and in liver and kidney and is a receptor for H-ferritin endocytosis. Journal of Experimental Medicine, 202, 955-965.
Clyde, K., Kyle, J. L., & Harris, E. (2006). Recent advances in deciphering viral and host determinants of dengue virus replication and pathogenesis. Journal of Virology, 80, 11418-11431.
Cohen, L. A., Gutierrez, L., Weiss, A., Leichtmann-Bardoogo, Y., Zhang, D.-l., Crooks, D. R., & Hentze, M. W. (2010). Serum ferritin is derived primarily from macrophages through a nonclassical secretory pathway. Blood, 116, 1574-1584.
Crişan, T. O., Plantinga, T. S., van de Veerdonk, F. L., Farcaş, M. F., Stoffels, M., Kullberg, B.-J., Netea, & M. G. (2011). Inflammasome-independent modulation of cytokine response by autophagy in human cells. PloS One, 6, e18666.
Falgout, B., Pethel, M., Zhang, Y., & Lai, C. (1991). Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. Journal of Virology, 65, 2467-2475.
Gonzalez, R., Jennings, L. L., Knuth, M., Orth, A. P., Klock, H. E., Ou, W., & Wang, Y. (2010). Screening the mammalian extracellular proteome for regulators of embryonic human stem cell pluripotency. Proceedings of The National Academy of Sciences, 107, 3552-3557.
Gray, C. P., Arosio, P., & Hersey, P. (2002). Heavy chain ferritin activates regulatory T cells by induction of changes in dendritic cells. Blood, 99, 3326-3334.
Green, S., Vaughn, D. W., Kalayanarooj, S., Nimmannitya, S., Suntayakorn, S., Nisalak, A., & Ennis, F. A. (1999). Elevated plasma interleukin-10 levels in acute dengue correlate with disease severity. Journal of Medical Virology, 59, 329-334.
Grom, A. A., Horne, A., & De Benedetti, F. (2016). Macrophage activation syndrome in the era of biologic therapy. Nature Reviews Rheumatology, 12(5), 259-268.
Halstead, S. B., Mahalingam, S., Marovich, M. A., Ubol, S., & Mosser, D. M. (2010). Intrinsic antibody-dependent enhancement of microbial infection in macrophages: disease regulation by immune complexes. The Lancet Infectious Diseases, 10, 712-722.
Harris, J. (2011). Autophagy and cytokines. Cytokine, 56(2), 140-144.
Henter, J. I., Horne, A., Aricó, M., Egeler, R. M., Filipovich, A. H., Imashuku, S., & Winiarski, J. (2007). HLH‐2004: diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. Pediatric Blood & Cancer, 48, 124-131.
Hober, D., Poli, L., Roblin, B., Gestas, P., Chungue, E., Granic, G., & Wattre, P. (1993). Serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) in dengue-infected patients. The American Journal of Tropical Medicine and Hygiene, 48, 324-331.
Hung, N. T., Lei, H.-Y., Lan, N. T., Lin, Y.-S., Huang, K.-J., Lien, L. B., & Huong, V. T. Q. (2004). Dengue hemorrhagic fever in infants: a study of clinical and cytokine profiles. The Journal of Infectious Diseases, 189, 221-232.
Idrees, S., & Ashfaq, U. A. (2012). A brief review on dengue molecular virology, diagnosis, treatment and prevalence in Pakistan. Genetic Vaccines and Therapy, 10, 6.
Janka, G. E. (2007). Familial and acquired hemophagocytic lymphohistiocytosis. European journal of Pediatrics, 166, 95-109.
Kenneth, N. S., Mudie, S., Naron, S., & Rocha, S. (2013). TfR1 interacts with the IKK complex and is involved in IKK–NF-κB signalling. Biochemical Journal, 449, 275-284.
Kimura, T., Jia, J., Kumar, S., Choi, S. W., Gu, Y., Mudd, M., & Farzam, F. (2017). Dedicated SNAREs and specialized TRIM cargo receptors mediate secretory autophagy. The EMBO Journal, 36, 42-60.
Kuchroo, V. K., Meyers, J. H., Umetsu, D. T., & DeKruyff, R. H. (2006). TIM family of genes in immunity and tolerance. Advances in Immunology, 91, 227-249.
Kuchroo, V. K., Umetsu, D. T., DeKruyff, R. H., & Freeman, G. J. (2003a). The TIM gene family: emerging roles in immunity and disease. Nature Reviews. Immunology, 3, 454.
Kuchroo, V. K., Umetsu, D. T., DeKruyff, R. H., & Freeman, G. J. (2003b). The TIM gene family: emerging roles in immunity and disease. Nature Reviews Immunology, 3, 454-462.
Kuno, G., & Bailey, R. E. (1994). Cytokine responses to dengue infection among Puerto Rican patients. Memórias do Instituto Oswaldo Cruz, 89, 179-182.
Lee, Y.-R., Hu, H.-Y., Kuo, S.-H., Lei, H.-Y., Lin, Y.-S., Yeh, T.-M., & Liu, H.-S. (2013). Dengue virus infection induces autophagy: an in vivo study. Journal of biomedical science, 20, 65.
Lee, Y.-R., Lei, H.-Y., Liu, M.-T., Wang, J.-R., Chen, S.-H., Jiang-Shieh, Y.-F., & Liu, H.-S. (2008). Autophagic machinery activated by dengue virus enhances virus replication. Virology, 374, 240-248.
Lei, H.-Y., Huang, K.-J., Lin, Y.-S., Yeh, T. M., Liu, H. S., & Liu, C.-C. (2008). Immunopathogenesis of dengue hemorrhagic fever. Am J Infect Dis, 4, 1-9.
Lei, H.-Y. (2009). Transient hemophagocytic activity in dengue immunopathogenesis. Journal of The Formosan Medical Association, 108, 595-598.
Leimberg, M. J., Prus, E., Konijn, A. M., & Fibach, E. (2008). Macrophages function as a ferritin iron source for cultured human erythroid precursors. Journal of Cellular Biochemistry, 103, 1211-1218.
Li, L., Fang, C. J., Ryan, J. C., Niemi, E. C., Lebrón, J. A., Björkman, P. J., & Nakamura, M. C. (2010). Binding and uptake of H-ferritin are mediated by human transferrin receptor-1. Proceedings of the National Academy of Sciences, 107, 3505-3510.
Li, R., Luo, C., Mines, M., Zhang, J., & Fan, G.-H. (2006). Chemokine CXCL12 induces binding of ferritin heavy chain to the chemokine receptor CXCR4, alters CXCR4 signaling, and induces phosphorylation and nuclear translocation of ferritin heavy chain. Journal of Biological Chemistry, 281, 37616-37627.
Luo, D., Xu, T., Hunke, C., Grüber, G., Vasudevan, S. G., & Lescar, J. (2008). Crystal structure of the NS3 protease-helicase from dengue virus. Journal of Virology, 82, 173-183.
Lv, S., Xu, Q., Sun, E., Yang, T., Li, J., Feng, Y., & Wu, D. (2015). Autophagy activated by bluetongue virus infection plays a positive role in its replication. Viruses, 7, 4657-4675.
Matusan, A. E., Pryor, M. J., Davidson, A. D., & Wright, P. J. (2001). Mutagenesis of the Dengue virus type 2 NS3 protein within and outside helicase motifs: effects on enzyme activity and virus replication. Journal of Virology, 75, 9633-9643.
Meertens, L., Carnec, X., Lecoin, M. P., Ramdasi, R., Guivel-Benhassine, F., Lew, E., & Amara, A. (2012). The TIM and TAM families of phosphatidylserine receptors mediate dengue virus entry. Cell Host & Microbe, 12, 544-557.
Morikawa, K., Oseko, F., & Morikawa, S. (1994). H-and L-rich ferritins suppress antibody production, but not proliferation, of human B lymphocytes in vitro. Blood, 83, 737-743.
Mohamed, W., Sethi, S., Darji, A., Mraheil, M. A., Hain, T., & Chakraborty, T. (2010). Antibody targeting the ferritin-like protein controls Listeria infection. Infection and Immunity, 78, 3306-3314.
Murray, M. T., White, K., & Munro, H. N. (1987). Conservation of ferritin heavy subunit gene structure: implications for the regulation of ferritin gene expression. Proceedings of the National Academy of Sciences, 84, 7438-7442.
Organization, W. H., Research, S. P. f., Diseases, T. i. T., Diseases, W. H. O. D. o. C. o. N. T., Epidemic, W. H. O., & Alert, P. (2009). Dengue: guidelines for diagnosis, treatment, prevention and control: World Health Organization.
Pérez, A. B., García, G., Sierra, B., Alvarez, M., Vázquez, S., Cabrera, M. V., & Denny, T. (2004). IL‐10 levels in dengue patients: some findings from the exceptional epidemiological conditions in Cuba. Journal of Medical Virology, 73, 230-234.
Park, H.-J., Lee, S. J., Kim, S.-H., Han, J., Bae, J., Kim, S. J., & Chun, T. (2011). IL-10 inhibits the starvation induced autophagy in macrophages via class I phosphatidylinositol 3-kinase (PI3K) pathway. Molecular Immunology, 48, 720-727.
Rothman, A. L., & Ennis, F. A. (1999). Immunopathogenesis of dengue hemorrhagic fever. Virology, 257, 1-6.
Puerta-Guardo, H., Mosso, C., Medina, F., Liprandi, F., Ludert, J. E., & del Angel, R. M. (2010). Antibody-dependent enhancement of dengue virus infection in U937 cells requires cholesterol-rich membrane microdomains. Journal of General Virology, 91, 394-403.
Recalcati, S., Invernizzi, P., Arosio, P., & Cairo, G. (2008). New functions for an iron storage protein: the role of ferritin in immunity and autoimmunity. Journal of Autoimmunity, 30, 84-89.
Ruddell, R. G., Hoang‐Le, D., Barwood, J. M., Rutherford, P. S., Piva, T. J., Watters, D. J., & Ramm, G. A. (2009). Ferritin functions as a proinflammatory cytokine via iron‐independent protein kinase C zeta/nuclear factor kappaB–regulated signaling in rat hepatic stellate cells. Hepatology, 49, 887-900.
Srichaikul, T., Punyagupta, S., Kanchanapoom, T., Chanokovat, C., Likittanasombat, K., & Leelasiri, A. (2008). Hemophagocytic syndrome in Dengue hemorrhagic fever with severe multiorgan complications. J Med Assoc Thai, 91, 104-109.
Tsai, T.-T., Chuang, Y.-J., Lin, Y.-S., Wan, S.-W., Chen, C.-L., & Lin, C.-F. (2013). An emerging role for the anti-inflammatory cytokine interleukin-10 in dengue virus infection. Journal of Biomedical Science, 20, 40.
Uchida, Y., Ke, B., Freitas, M. C. S., Ji, H., Zhao, D., Benjamin, E. R., & Busuttil, R. W. (2010). The emerging role of T cell immunoglobulin mucin‐1 in the mechanism of liver ischemia and reperfusion injury in the mouse. Hepatology, 51, 1363-1372.
van de Weg, C. A., Huits, R. M., Pannuti, C. S., Brouns, R. M., van den Berg, R. W., van den Ham, H.-J., & Meijers, J. C. (2014). Hyperferritinaemia in dengue virus infected patients is associated with immune activation and coagulation disturbances. PLoS Neglected Tropical Diseases, 8, e3214.
Wan, S.-W., Lin, C.-F., Yeh, T.-M., Liu, C.-C., Liu, H.-S., Wang, S., & Lin, Y.-S. (2013). Autoimmunity in dengue pathogenesis. Journal of The Formosan Medical Association, 112, 3-11.
Wang, S.-F., Wang, W.-H., Chang, K., Chen, Y.-H., Tseng, S.-P., Yen, C.-H., & Chen, Y.-M. A. (2016). Severe dengue fever outbreak in Taiwan. The American Journal of Tropical Medicine and Hygiene, 94, 193-197.
Wu, T.-t., Li, W.-M., & Yao, Y.-M. (2016). Interactions between autophagy and inhibitory cytokines. International Journal of Biological Sciences, 12, 884.
Weiss, G. (2005). Modification of iron regulation by the inflammatory response. Best Practice & Research Clinical Haematology, 18, 183-201.

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