自由營生棘阿米巴原蟲是相當常見的一種原生動物。它感染眼睛時，會造成棘阿米巴角膜炎；而感染腦部及脊椎時，會造成肉芽腫性腦炎。受到棘阿米巴感染後，患者無法發展出良好的記憶性免疫，因而常出現重複感染和復發，所以我們推測先天性免疫對於對抗棘阿米巴的感染占很重要的角色。已知人類單核球參與對抗棘阿米巴的感染，而且棘阿米巴分泌物，會透過刺激人類單核球/巨噬細胞產生大量的促發炎激素和抑制發炎激素，來調控人類的免疫反應，但其中確切的機制仍不清楚。本論文的研究方向分為兩個部分，第一，探討棘阿米巴分泌物刺激人類單核球THP-1細胞產生IL-10的訊息傳遞路徑；第二，研究棘阿米巴透過甚麼方式，來調控人類的發炎反應。當培養棘阿米巴的培養液 (aCM)刺激THP-1細胞後，磷酸化蛋白激酶A (p-PKA)與磷酸化cAMP反應元件結合蛋白 (p-CREB)兩者的蛋白質表達量會陸續提升，同時介白素-10 (IL-10)的mRNA表達量也隨之大幅上升。這表示aCM刺激THP-1細胞後，THP-1細胞會透過活化PKA的訊息傳導路徑，使得增加THP-1細胞中IL-10的轉錄表達量。除此之外，由棘阿米巴分泌物中純化出來的棘阿米巴分泌蛋白質 (Asp)，同樣可以刺激THP-1細胞提高促發炎細胞激素 (IL-12和IL-6)和抑制發炎細胞激素 (IL-10)的mRNA表達量。綜合本研究成果顯示棘阿米巴分泌物中的Asp有調控人類發炎反應的能力，它可能是棘阿米巴逃離人類免疫反應攻擊的重要工具，未來若能更進一步的確實找到作用蛋白質及其機制，便有望做為藥物開發的標的。

Free-living amoeba of the pathogenic Acanthamoeba species causes chronic diseases in humans. Reinfection and recurrence in Acanthamoeba patients suggest that immunological memory is not well developed. The innate immune apparatus is crucial for the resolution of the disease. Previous studies have shown that monocytes/macrophages are involved in controlling Acanthamoeba infections. When THP-1 cells (human monocytic cells) were stimulated with Acanthamoeba extracellular components, proinflammatory cytokines and anti-inflammatory cytokines production were enhanced. However, the effect of Acanthamoeba infection in modulating a human immune response remains unclear. In this study, the results showed that the phosphorylation of protein kinase A (PKA) and cAMP response element-binding protein (CREB) were enhanced in THP-1 cells with Acanthamoeba extracellular components. Meanwhile, the transcriptional activity of the interleuukin-10 (IL-10) gene, an anti-inflammatory cytokine, was significantly augmented. This suggests that Acanthamoeba extracellular components enhanced the IL-10 gene expression through the PKA-dependent pathway. In addition, we isolated Acanthamoeba secreted proteins (Asp) from Acanthamoeba extracellular components. The Asp not only enhanced the expression of proinflammatory cytokines including IL-12 and IL-6, but also induced IL-10 in THP-1 cells. In conclusion, the extracellular proteins of Acanthamoeba can modulate inflammation responses in human innate immune cells, which might both promote both the immune evasion of Acanthamoeba and limit the inflammatory responses.

Alizadeh, H., S. Apte, M. S. El-Agha, L. Li, M. Hurt, K. Howard, H. D. Cavanagh, J. P. McCulley and J. Y. Niederkorn (2001). "Tear IgA and serum IgG antibodies against Acanthamoeba in patients with Acanthamoeba keratitis." Cornea 20(6): 622-627.

Beavo, J. A. and L. L. Brunton (2002). "Cyclic nucleotide research -- still expanding after half a century." Nat Rev Mol Cell Biol 3(9): 710-718.

Biron, C. A. and R. T. Gazzinelli (1995). "Effects of IL-12 on immune responses to microbial infections: a key mediator in regulating disease outcome." Curr Opin Immunol 7(4): 485-496.

Brenner, S., S. Prosch, K. Schenke-Layland, U. Riese, U. Gausmann and C. Platzer (2003). "cAMP-induced Interleukin-10 promoter activation depends on CCAAT/enhancer-binding protein expression and monocytic differentiation." J Biol Chem 278(8): 5597-5604.

Brossart, P., F. Grunebach, G. Stuhler, V. L. Reichardt, R. Mohle, L. Kanz and W. Brugger (1998). "Generation of functional human dendritic cells from adherent peripheral blood monocytes by CD40 ligation in the absence of granulocyte-macrophage colony-stimulating factor." Blood 92(11): 4238-4247.

Bystrom, J., I. Evans, J. Newson, M. Stables, I. Toor, N. van Rooijen, M. Crawford, P. Colville-Nash, S. Farrow and D. W. Gilroy (2008). "Resolution-phase macrophages possess a unique inflammatory phenotype that is controlled by cAMP." Blood 112(10): 4117-4127.

Castell, J. V., M. J. Gomez-Lechon, M. David, T. Hirano, T. Kishimoto and P. C. Heinrich (1988). "Recombinant human interleukin-6 (IL-6/BSF-2/HSF) regulates the synthesis of acute phase proteins in human hepatocytes." FEBS Lett 232(2): 347-350.

Castellani, A. (1930). "An amoeba found in culture of yeast: preliminary note." J Trop Med Hyg 33: 160.

Chomarat, P., J. Banchereau, J. Davoust and A. K. Palucka (2000). "IL-6 switches the differentiation of monocytes from dendritic cells to macrophages." Nat Immunol 1(6): 510-514.

Clarke, D. W. and J. Y. Niederkorn (2006). "The immunobiology of Acanthamoeba keratitis." Microbes Infect 8(5): 1400-1405.

Couper, K. N., D. G. Blount and E. M. Riley (2008). "IL-10: the master regulator of immunity to infection." J Immunol 180(9): 5771-5777.

Cursons, R. T., T. J. Brown, E. A. Keys, K. M. Moriarty and D. Till (1980). "Immunity to pathogenic free-living amoebae: role of humoral antibody." Infect Immun 29(2): 401-407.

Delghandi, M. P., M. Johannessen and U. Moens (2005). "The cAMP signalling pathway activates CREB through PKA, p38 and MSK1 in NIH 3T3 cells." Cell Signal 17(11): 1343-1351.

Elliott, D. E., J. Li, A. Blum, A. Metwali, K. Qadir, J. F. Urban, Jr. and J. V. Weinstock (2003). "Exposure to schistosome eggs protects mice from TNBS-induced colitis." Am J Physiol Gastrointest Liver Physiol 284(3): G385-391.

Finlay, C. M., K. P. Walsh and K. H. Mills (2014). "Induction of regulatory cells by helminth parasites: exploitation for the treatment of inflammatory diseases." Immunol Rev 259(1): 206-230.

Flynn, R. J. and G. Mulcahy (2008). "The roles of IL-10 and TGF-beta in controlling IL-4 and IFN-gamma production during experimental Fasciola hepatica infection." Int J Parasitol 38(14): 1673-1680.

Fuerst, P. A., G. C. Booton and M. Crary (2015). "Phylogenetic analysis and the evolution of the 18S rRNA gene typing system of Acanthamoeba." J Eukaryot Microbiol 62(1): 69-84.

Harnett, M. M., A. J. Melendez and W. Harnett (2010). "The therapeutic potential of the filarial nematode-derived immunodulator, ES-62 in inflammatory disease." Clin Exp Immunol 159(3): 256-267.

Hasko, G. and P. Pacher (2012). "Regulation of macrophage function by adenosine." Arterioscler Thromb Vasc Biol 32(4): 865-869.

Hertz, A. L. and J. A. Beavo (2011). "Cyclic nucleotides and phosphodiesterases in monocytic differentiation." Handb Exp Pharmacol(204): 365-390.

Hilbert, D. M., M. P. Cancro, P. A. Scherle, R. P. Nordan, J. Van Snick, W. Gerhard and S. Rudikoff (1989). "T cell derived IL-6 is differentially required for antigen-specific antibody secretion by primary and secondary B cells." J Immunol 143(12): 4019-4024.

Hofer, A. M. and K. Lefkimmiatis (2007). "Extracellular calcium and cAMP: second messengers as "third messengers"?" Physiology (Bethesda) 22: 320-327.

Huang, J. M., W. C. Lin, S. C. Li, M. H. Shih, W. C. Chan, J. W. Shin and F. C. Huang (2016). "Comparative proteomic analysis of extracellular secreted proteins expressed by two pathogenic Acanthamoeba castellanii clinical isolates and a non-pathogenic ATCC strain." Exp Parasitol 166: 60-67.

Hubner, M. P., L. E. Layland and A. Hoerauf (2013). "Helminths and their implication in sepsis - a new branch of their immunomodulatory behaviour?" Pathog Dis 69(2): 127-141.
Hurt, M., V. Proy, J. Y. Niederkorn and H. Alizadeh (2003). "The interaction of Acanthamoeba castellanii cysts with macrophages and neutrophils." J Parasitol 89(3): 565-572.

Jones, K. R. and E. M. Kang (2015). "Graft versus host disease: New insights into A2A receptor agonist therapy." Comput Struct Biotechnol J 13: 101-105.

Köhsler, M., M. Mrva and J. Walochnik (2016). Acanthamoeba. Molecular Parasitology: Protozoan Parasites and their Molecules. J. Walochnik and M. Duchêne. Vienna, Springer Vienna: 285-324.

Kamenetsky, M., S. Middelhaufe, E. M. Bank, L. R. Levin, J. Buck and C. Steegborn (2006). "Molecular details of cAMP generation in mammalian cells: a tale of two systems." J Mol Biol 362(4): 623-639.

Khan, N. A. (2006). "Acanthamoeba: biology and increasing importance in human health." FEMS Microbiol Rev 30(4): 564-595.

Kim, H. P. and W. J. Leonard (2007). "CREB/ATF-dependent T cell receptor-induced FoxP3 gene expression: a role for DNA methylation." J Exp Med 204(7): 1543-1551.

Landry, Y., N. Niederhoffer, E. Sick and J. P. Gies (2006). "Heptahelical and other G-protein-coupled receptors (GPCRs) signaling." Curr Med Chem 13(1): 51-63.
Martinez, A. J. (1991). "Infection of the central nervous system due to Acanthamoeba." Rev Infect Dis 13 Suppl 5: S399-402.

Martinez, A. J. and G. S. Visvesvara (1997). "Free-living, amphizoic and opportunistic amebas." Brain Pathol 7(1): 583-598.

Mattana, A., M. Sanna, A. Cano, G. Delogu, G. Erre, C. W. Roberts, F. L. Henriquez, P. L. Fiori and P. Cappuccinelli (2016). "Acanthamoeba castellanii Genotype T4 Stimulates the Production of Interleukin-10 as Well as Proinflammatory Cytokines in THP-1 Cells, Human Peripheral Blood Mononuclear Cells, and Human Monocyte-Derived Macrophages." Infect Immun 84(10): 2953-2962.

Mayr, B. and M. Montminy (2001). "Transcriptional regulation by the phosphorylation-dependent factor CREB." Nat Rev Mol Cell Biol 2(8): 599-609.

McInnes, I. B., B. P. Leung, M. Harnett, J. A. Gracie, F. Y. Liew and W. Harnett (2003). "A Novel Therapeutic Approach Targeting Articular Inflammation Using the Filarial Nematode-Derived Phosphorylcholine-Containing Glycoprotein ES-62." The Journal of Immunology 171(4): 2127-2133.

Moore, K. W., R. de Waal Malefyt, R. L. Coffman and A. O'Garra (2001). "Interleukin-10 and the interleukin-10 receptor." Annu Rev Immunol 19: 683-765.

Najar, H. M., S. Ruhl, A. C. Bru-Capdeville and J. H. Peters (1990). "Adenosine and its derivatives control human monocyte differentiation into highly accessory cells versus macrophages." J Leukoc Biol 47(5): 429-439.

Neelam, S. and J. Y. Niederkorn (2017). "Pathobiology and Immunobiology of Acanthamoeba Keratitis: Insights from Animal Models." Yale J Biol Med 90(2): 261-268.

Niederkorn, J. Y. (2002). "The role of the innate and adaptive immune responses in Acanthamoeba keratitis." Arch Immunol Ther Exp (Warsz) 50(1): 53-59.

Niederkorn, J. Y., H. Alizadeh, H. Leher, S. Apte, S. El Agha, L. Ling, M. Hurt, K. Howard, H. D. Cavanagh and J. P. McCulley (2002). "Role of tear anti-acanthamoeba IgA in Acanthamoeba keratitis." Adv Exp Med Biol 506(Pt B): 845-850.

Niederkorn, J. Y., H. Alizadeh, H. Leher and J. P. McCulley (1999). "The pathogenesis of Acanthamoeba keratitis." Microbes Infect 1(6): 437-443.

Panjwani, N. (2010). "Pathogenesis of acanthamoeba keratitis." Ocul Surf 8(2): 70-79.
Raker, V. K., C. Becker and K. Steinbrink (2016). "The cAMP Pathway as Therapeutic Target in Autoimmune and Inflammatory Diseases." Frontiers in Immunology 7: 123.

Randolph, G. J., S. Beaulieu, S. Lebecque, R. M. Steinman and W. A. Muller (1998). "Differentiation of monocytes into dendritic cells in a model of transendothelial trafficking." Science 282(5388): 480-483.

Sciaraffia, E., A. Riccomi, R. Lindstedt, V. Gesa, E. Cirelli, M. Patrizio, M. T. De Magistris and S. Vendetti (2014). "Human monocytes respond to extracellular cAMP through A2A and A2B adenosine receptors." J Leukoc Biol 96(1): 113-122.

Serezani, C. H., M. N. Ballinger, D. M. Aronoff and M. Peters-Golden (2008). "Cyclic AMP: master regulator of innate immune cell function." Am J Respir Cell Mol Biol 39(2): 127-132.

Sewell, D., Z. Qing, E. Reinke, D. Elliot, J. Weinstock, M. Sandor and Z. Fabry (2003). "Immunomodulation of experimental autoimmune encephalomyelitis by helminth ova immunization." Int Immunol 15(1): 59-69.

Siddiqui, R. and N. A. Khan (2012). "Biology and pathogenesis of Acanthamoeba." Parasit Vectors 5: 6.

Stewart, G. L., I. Kim, K. Shupe, H. Alizadeh, R. Silvany, J. P. McCulley and J. Y. Niederkorn (1992). "Chemotactic response of macrophages to Acanthamoeba castellanii antigen and antibody-dependent macrophage-mediated killing of the parasite." J Parasitol 78(5): 849-855.

Sunahara, R. K. and R. Taussig (2002). "Isoforms of mammalian adenylyl cyclase: multiplicities of signaling." Mol Interv 2(3): 168-184.

Thebpatiphat, N., K. M. Hammersmith, F. N. Rocha, C. J. Rapuano, B. D. Ayres, P. R. Laibson, R. C. Eagle, Jr. and E. J. Cohen (2007). "Acanthamoeba keratitis: a parasite on the rise." Cornea 26(6): 701-706.

Tormo, A. J., M. C. Letellier, M. Sharma, G. Elson, S. Crabe and J. F. Gauchat (2012). "IL-6 activates STAT5 in T cells." Cytokine 60(2): 575-582.

Walochnik, J., A. Obwaller, E. M. Haller-Schober and H. Aspock (2001). "Anti-Acanthamoeba IgG, IgM, and IgA immunoreactivities in correlation to strain pathogenicity." Parasitol Res 87(8): 651-656.

Wen, A. Y., K. M. Sakamoto and L. S. Miller (2010). "The role of the transcription factor CREB in immune function." J Immunol 185(11): 6413-6419.

Xu, X. J., J. S. Reichner, B. Mastrofrancesco, W. L. Henry, Jr. and J. E. Albina (2008). "Prostaglandin E2 suppresses lipopolysaccharide-stimulated IFN-beta production." J Immunol 180(4): 2125-2131.

Yang, B. C., Y. S. Wang, L. C. Lin and M. F. Liu (1997). "Induction of apoptosis and cytokine gene expression in T-cell lines by sera of patients with systemic lupus erythematosus." Scand J Immunol 45(1): 96-102.

Zaccone, P., O. Burton, N. Miller, F. M. Jones, D. W. Dunne and A. Cooke (2009). "Schistosoma mansoni egg antigens induce Treg that participate in diabetes prevention in NOD mice." Eur J Immunol 39(4): 1098-1107.