摘要
類鐸受體9經由辨識致病性CpG去氧核醣核酸(CpG DNA)/CpG寡聚核苷酸(CpG ODN)會產生促發炎因子。在這過程中，短片段CpG寡聚核苷酸(CpG ODN)進入核內體以及類鐸受體9從內質網運輸至核內體則是引起初級免疫反應的兩個重要步驟。而ADP核糖基化因子(ARF)是屬於與鳥嘌呤核苷酸結合的Ras蛋白，主要參與細胞中的胞飲作用和囊泡運輸。在本研究中，我們發現抑制ARF3或ARF6會降低CpG ODN所調控的反應，而持續活化ARF3或ARF6則會提高此反應。進一步的結果指出在ARF6活化的細胞中會增加CpG ODN被攝入細胞，但於ARF6缺失的細胞中則會減少CpG ODN被攝入細胞的結果。細胞預先施與CpG ODN則會誘發ARF6的活性進而增加CpG ODN被攝入細胞；此外並證明Class III PI3K為下游ARF6調控CpG ODN進入細胞所需。這些結果推得出一條以class III PI3K-ARF6為軸的路徑，此路徑經由調控CpG ODN進入細胞的過程，從而調節類鐸受體9的訊息。相對於ARF6參與CpG ODN/類鐸受體9所調控的反應，ARF3的活化則不影響CpG ODN進入細胞，但是參與類鐸受體9運輸至核內體的過程，也因此影響了裂解型式的功能性類鐸受體9形成。而於免疫沉澱的實驗中則指出具活性的ARF3會與類鐸受體9以及受體的運送蛋白UNC93B1相互作用，所以證明ARF3是經由調控類鐸受體9從內質網運輸至核內體的過程，進而調控類鐸受體9的訊息。總結來說，class I ARF3和class III ARF6在類鐸受體9的訊息路徑中各自扮演不同的角色，ARF6參與CpG ODN進入細胞內的過程，而ARF3則調控類鐸受體9的運輸從而活化CpG ODN/類鐸受體9的免疫反應。

Abstract
Toll-like receptor 9 (TLR9) is responsible for proinflammatory cytokine production via recognizing pathogenic unmethylated CpG DNA. In the process, CpG oligodeoxynucleotides (CpG ODN) uptake into endosomes and TLR9 trafficking from endoplasmic reticulum (ER) to endosomes are two critical steps in eliciting innate immune responses. ADP-ribosylation factors (ARFs) are members of the Ras superfamily of guanine nucleotide-binding proteins, which are involved in mediating a wide variety of cellular events including endocytosis and vesicle trafficking. Here, we found that inhibition of ARF3 or ARF6 by dominant mutants and siRNA impaired CpG ODN-mediated responses, whereas constitutive active ARF3 or ARF6 mutant enhanced these responses. Additional studies showed that CpG ODN uptake was increased in ARF6-activated cells but impaired in ARF6-defective cells. Cells pre-treated with CpG ODN had increased CpG ODN uptake due to CpG ODN-induced ARF6 activity. Further studies with ARF6-defective and ARF6-activated cells demonstrated that class III phosphatidylinositol 3-kinases (PI3K) were required for downstream ARF6 regulation of CpG ODN uptake. These results propose that a novel class III PI3K-ARF6 axis pathway mediates TLR9 signaling by regulating the cellular uptake of CpG ODN. In contrast to the involvement of ARF6 in CpG ODN/TLR9-mediated responses, ARF3 activity is essential for TLR9 trafficking into endolysosomes without affecting CpG ODN uptake, thereby regulating the formation of functional cleaved TLR9. In addition, experiment with immnuoprecipitation indicated that activation of ARF3 interacted with the TLR9 and the carrier protein, UNC93B1. Thus, the results demonstrate that ARF3 mediates CpG ODN-driven responses by regulating TLR9 trafficking from ER into endolysosomes. Collectively, class I ARF3 and class III ARF6 play distinct roles in CpG ODN/TLR9-mediated responses. ARF6 directly participates in CpG ODN internalization, while ARF3 drives TLR9 trafficking to activate CpG ODN/TLR9-induced immune responses.

REFERENCES
1. Janeway, C. A., Jr., and Medzhitov, R. (1998) Introduction: the role of innate immunity in the adaptive immune response. Semin Immunol 10, 349-350
2. Janeway, C. A., Jr., and Medzhitov, R. (2002) Innate immune recognition. Annu Rev Immunol 20, 197-216
3. Akira, S., Takeda, K., and Kaisho, T. (2001) Toll-like receptors: critical proteins linking innate and acquired immunity. Nat Immunol 2, 675-680
4. McGettrick, A. F., and O'Neill, L. A. (2010) Localisation and trafficking of Toll-like receptors: an important mode of regulation. Curr Opin Immunol 22, 20-27
5. Kawai, T., and Akira, S. (2006) TLR signaling. Cell death and differentiation 13, 816-825
6. Akira, S., Uematsu, S., and Takeuchi, O. (2006) Pathogen recognition and innate immunity. Cell 124, 783-801
7. O'Neill, L. A., Golenbock, D., and Bowie, A. G. (2013) The history of Toll-like receptors - redefining innate immunity. Nature reviews. Immunology 13, 453-460
8. Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., Matsumoto, M., Hoshino, K., Wagner, H., Takeda, K., and Akira, S. (2000) A Toll-like receptor recognizes bacterial DNA. Nature 408, 740-745
9. Bauer, S., Kirschning, C. J., Hacker, H., Redecke, V., Hausmann, S., Akira, S., Wagner, H., and Lipford, G. B. (2001) Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci U S A 98, 9237-9242
10. Lund, J., Sato, A., Akira, S., Medzhitov, R., and Iwasaki, A. (2003) Toll-like receptor 9-mediated recognition of Herpes simplex virus-2 by plasmacytoid dendritic cells. J Exp Med 198, 513-520
11. Krug, A., French, A. R., Barchet, W., Fischer, J. A., Dzionek, A., Pingel, J. T., Orihuela, M. M., Akira, S., Yokoyama, W. M., and Colonna, M. (2004) TLR9-dependent recognition of MCMV by IPC and DC generates coordinated cytokine responses that activate antiviral NK cell function. Immunity 21, 107-119
12. Hacker, H., Mischak, H., Miethke, T., Liptay, S., Schmid, R., Sparwasser, T., Heeg, K., Lipford, G. B., and Wagner, H. (1998) CpG-DNA-specific activation of antigen-presenting cells requires stress kinase activity and is preceded by non-specific endocytosis and endosomal maturation. Embo J 17, 6230-6240
13. Hacker, H., Vabulas, R. M., Takeuchi, O., Hoshino, K., Akira, S., and Wagner, H. (2000) Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6. J Exp Med 192, 595-600
14. Akira, S., and Hemmi, H. (2003) Recognition of pathogen-associated molecular patterns by TLR family. Immunol Lett 85, 85-95
15. Kobayashi, T., Walsh, M. C., and Choi, Y. (2004) The role of TRAF6 in signal transduction and the immune response. Microbes Infect 6, 1333-1338
16. Vilaysane, A., and Muruve, D. A. (2009) The innate immune response to DNA. Semin Immunol 21, 208-214
17. Krieg, A. M. (2002) CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 20, 709-760
18. Kumagai, Y., Takeuchi, O., and Akira, S. (2008) TLR9 as a key receptor for the recognition of DNA. Adv Drug Deliv Rev 60, 795-804
19. Gilliet, M., Cao, W., and Liu, Y. J. (2008) Plasmacytoid dendritic cells: sensing nucleic acids in viral infection and autoimmune diseases. Nature reviews. Immunology 8, 594-606
20. Kuo, C. C., Liang, S. M., and Liang, C. M. (2006) CpG-B oligodeoxynucleotide promotes cell survival via up-regulation of Hsp70 to increase Bcl-xL and to decrease apoptosis-inducing factor translocation. J Biol Chem 281, 38200-38207
21. Kuo, C. C., Liang, C. M., Lai, C. Y., and Liang, S. M. (2007) Involvement of heat shock protein (Hsp)90 beta but not Hsp90 alpha in antiapoptotic effect of CpG-B oligodeoxynucleotide. J Immunol 178, 6100-6108
22. Latz, E., Schoenemeyer, A., Visintin, A., Fitzgerald, K. A., Monks, B. G., Knetter, C. F., Lien, E., Nilsen, N. J., Espevik, T., and Golenbock, D. T. (2004) TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat Immunol 5, 190-198
23. Takeshita, F., Gursel, I., Ishii, K. J., Suzuki, K., Gursel, M., and Klinman, D. M. (2004) Signal transduction pathways mediated by the interaction of CpG DNA with Toll-like receptor 9. Semin Immunol 16, 17-22
24. Kuo, C. C., Lin, W. T., Liang, C. M., and Liang, S. M. (2006) Class I and III Phosphatidylinositol 3'-Kinase Play Distinct Roles in TLR Signaling Pathway. J Immunol 176, 5943-5949
25. Park, B., Buti, L., Lee, S., Matsuwaki, T., Spooner, E., Brinkmann, M. M., Nishihara, M., and Ploegh, H. L. (2011) Granulin is a soluble cofactor for toll-like receptor 9 signaling. Immunity 34, 505-513
26. Marshak-Rothstein, A. (2006) Toll-like receptors in systemic autoimmune disease. Nature reviews. Immunology 6, 823-835
27. Christensen, S. R., and Shlomchik, M. J. (2007) Regulation of lupus-related autoantibody production and clinical disease by Toll-like receptors. Semin Immunol 19, 11-23
28. Leifer, C. A., Kennedy, M. N., Mazzoni, A., Lee, C., Kruhlak, M. J., and Segal, D. M. (2004) TLR9 is localized in the endoplasmic reticulum prior to stimulation. J Immunol 173, 1179-1183
29. Chockalingam, A., Brooks, J. C., Cameron, J. L., Blum, L. K., and Leifer, C. A. (2009) TLR9 traffics through the Golgi complex to localize to endolysosomes and respond to CpG DNA. Immunol Cell Biol 87, 209-217
30. Brinkmann, M. M., Spooner, E., Hoebe, K., Beutler, B., Ploegh, H. L., and Kim, Y. M. (2007) The interaction between the ER membrane protein UNC93B and TLR3, 7, and 9 is crucial for TLR signaling. The Journal of cell biology 177, 265-275
31. Kim, Y. M., Brinkmann, M. M., Paquet, M. E., and Ploegh, H. L. (2008) UNC93B1 delivers nucleotide-sensing toll-like receptors to endolysosomes. Nature 452, 234-238
32. D'Souza-Schorey, C., and Chavrier, P. (2006) ARF proteins: roles in membrane traffic and beyond. Nat Rev Mol Cell Biol 7, 347-358
33. Inoue, H., and Randazzo, P. A. (2007) Arf GAPs and their interacting proteins. Traffic 8, 1465-1475
34. Randazzo, P. A., Inoue, H., and Bharti, S. (2007) Arf GAPs as regulators of the actin cytoskeleton. Biol Cell 99, 583-600
35. Manolea, F., Chun, J., Chen, D. W., Clarke, I., Summerfeldt, N., Dacks, J. B., and Melancon, P. (2010) Arf3 is activated uniquely at the trans-Golgi network by brefeldin A-inhibited guanine nucleotide exchange factors. Molecular biology of the cell 21, 1836-1849
36. Brown, H. A., Gutowski, S., Moomaw, C. R., Slaughter, C., and Sternweis, P. C. (1993) ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity. Cell 75, 1137-1144
37. Cockcroft, S., Thomas, G. M., Fensome, A., Geny, B., Cunningham, E., Gout, I., Hiles, I., Totty, N. F., Truong, O., and Hsuan, J. J. (1994) Phospholipase D: a downstream effector of ARF in granulocytes. Science 263, 523-526
38. Honda, A., Nogami, M., Yokozeki, T., Yamazaki, M., Nakamura, H., Watanabe, H., Kawamoto, K., Nakayama, K., Morris, A. J., Frohman, M. A., and Kanaho, Y. (1999) Phosphatidylinositol 4-phosphate 5-kinase alpha is a downstream effector of the small G protein ARF6 in membrane ruffle formation. Cell 99, 521-532
39. Krauss, M., Kinuta, M., Wenk, M. R., De Camilli, P., Takei, K., and Haucke, V. (2003) ARF6 stimulates clathrin/AP-2 recruitment to synaptic membranes by activating phosphatidylinositol phosphate kinase type Igamma. The Journal of cell biology 162, 113-124
40. Kawai, T., and Akira, S. (2010) The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat Immunol 11, 373-384
41. Messina, J. P., Gilkeson, G. S., and Pisetsky, D. S. (1991) Stimulation of in vitro murine lymphocyte proliferation by bacterial DNA. J Immunol 147, 1759-1764
42. Halpern, M. D., Kurlander, R. J., and Pisetsky, D. S. (1996) Bacterial DNA induces murine interferon-gamma production by stimulation of interleukin-12 and tumor necrosis factor-alpha. Cell Immunol 167, 72-78
43. Yi, A. K., and Krieg, A. M. (1998) CpG DNA rescue from anti-IgM-induced WEHI-231 B lymphoma apoptosis via modulation of I kappa B alpha and I kappa B beta and sustained activation of nuclear factor-kappa B/c-Rel. J Immunol 160, 1240-1245
44. Tokunaga, T., Yamamoto, H., Shimada, S., Abe, H., Fukuda, T., Fujisawa, Y., Furutani, Y., Yano, O., Kataoka, T., Sudo, T., and et al. (1984) Antitumor activity of deoxyribonucleic acid fraction from Mycobacterium bovis BCG. I. Isolation, physicochemical characterization, and antitumor activity. J Natl Cancer Inst 72, 955-962
45. Yamamoto, S., Kuramoto, E., Shimada, S., and Tokunaga, T. (1988) In vitro augmentation of natural killer cell activity and production of interferon-alpha/beta and -gamma with deoxyribonucleic acid fraction from Mycobacterium bovis BCG. Jpn J Cancer Res 79, 866-873
46. Stacey, K. J., Sweet, M. J., and Hume, D. A. (1996) Macrophages ingest and are activated by bacterial DNA. J Immunol 157, 2116-2122
47. Yi, A. K., Tuetken, R., Redford, T., Waldschmidt, M., Kirsch, J., and Krieg, A. M. (1998) CpG motifs in bacterial DNA activate leukocytes through the pH-dependent generation of reactive oxygen species. J Immunol 160, 4755-4761
48. Krieg, A. M., Yi, A. K., Matson, S., Waldschmidt, T. J., Bishop, G. A., Teasdale, R., Koretzky, G. A., and Klinman, D. M. (1995) CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 374, 546-549
49. Kline, J. N., Waldschmidt, T. J., Businga, T. R., Lemish, J. E., Weinstock, J. V., Thorne, P. S., and Krieg, A. M. (1998) Modulation of airway inflammation by CpG oligodeoxynucleotides in a murine model of asthma. J Immunol 160, 2555-2559
50. Shirota, H., Sano, K., Hirasawa, N., Terui, T., Ohuchi, K., Hattori, T., Shirato, K., and Tamura, G. (2001) Novel roles of CpG oligodeoxynucleotides as a leader for the sampling and presentation of CpG-tagged antigen by dendritic cells. J Immunol 167, 66-74
51. Takeda, K., and Akira, S. (2001) Roles of Toll-like receptors in innate immune responses. Genes Cells 6, 733-742
52. Krieg, A. M. (2007) Development of TLR9 agonists for cancer therapy. J Clin Invest 117, 1184-1194
53. Grant, B. D., and Donaldson, J. G. (2009) Pathways and mechanisms of endocytic recycling. Nat Rev Mol Cell Biol 10, 597-608
54. Burns, K., Martinon, F., Esslinger, C., Pahl, H., Schneider, P., Bodmer, J. L., Di Marco, F., French, L., and Tschopp, J. (1998) MyD88, an adapter protein involved in interleukin-1 signaling. J Biol Chem 273, 12203-12209
55. Ewald, S. E., Lee, B. L., Lau, L., Wickliffe, K. E., Shi, G. P., Chapman, H. A., and Barton, G. M. (2008) The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor. Nature 456, 658-662
56. Park, B., Brinkmann, M. M., Spooner, E., Lee, C. C., Kim, Y. M., and Ploegh, H. L. (2008) Proteolytic cleavage in an endolysosomal compartment is required for activation of Toll-like receptor 9. Nat Immunol 9, 1407-1414
57. Stein, M. P., Feng, Y., Cooper, K. L., Welford, A. M., and Wandinger-Ness, A. (2003) Human VPS34 and p150 are Rab7 interacting partners. Traffic 4, 754-771
58. Kuo, C. C., Kuo, C. W., Liang, C. M., and Liang, S. M. (2005) A transcriptomic and proteomic analysis of the effect of CpG-ODN on human THP-1 monocytic leukemia cells. Proteomics 5, 894-906
59. Sivori, S., Falco, M., Carlomagno, S., Romeo, E., Soldani, C., Bensussan, A., Viola, A., Moretta, L., and Moretta, A. (2010) A novel KIR-associated function: evidence that CpG DNA uptake and shuttling to early endosomes is mediated by KIR3DL2. Blood 116, 1637-1647
60. Butler, M., Crooke, R. M., Graham, M. J., Lemonidis, K. M., Lougheed, M., Murray, S. F., Witchell, D., Steinbrecher, U., and Bennett, C. F. (2000) Phosphorothioate oligodeoxynucleotides distribute similarly in class A scavenger receptor knockout and wild-type mice. J Pharmacol Exp Ther 292, 489-496
61. Gursel, M., Gursel, I., Mostowski, H. S., and Klinman, D. M. (2006) CXCL16 influences the nature and specificity of CpG-induced immune activation. J Immunol 177, 1575-1580
62. Anderson, R. B., Cianciolo, G. J., Kennedy, M. N., and Pizzo, S. V. (2008) Alpha 2-macroglobulin binds CpG oligodeoxynucleotides and enhances their immunostimulatory properties by a receptor-dependent mechanism. J Leukoc Biol 83, 381-392
63. Tabeta, K., Hoebe, K., Janssen, E. M., Du, X., Georgel, P., Crozat, K., Mudd, S., Mann, N., Sovath, S., Goode, J., Shamel, L., Herskovits, A. A., Portnoy, D. A., Cooke, M., Tarantino, L. M., Wiltshire, T., Steinberg, B. E., Grinstein, S., and Beutler, B. (2006) The Unc93b1 mutation 3d disrupts exogenous antigen presentation and signaling via Toll-like receptors 3, 7 and 9. Nat Immunol 7, 156-164
64. Zanetti, G., Pahuja, K. B., Studer, S., Shim, S., and Schekman, R. (2012) COPII and the regulation of protein sorting in mammals. Nature cell biology 14, 20-28
65. Lee, B. L., Moon, J. E., Shu, J. H., Yuan, L., Newman, Z. R., Schekman, R., and Barton, G. M. (2013) UNC93B1 mediates differential trafficking of endosomal TLRs. eLife 2, e00291
66. Kondo, Y., Hanai, A., Nakai, W., Katoh, Y., Nakayama, K., and Shin, H. W. (2012) ARF1 and ARF3 are required for the integrity of recycling endosomes and the recycling pathway. Cell structure and function 37, 141-154
67. Wu, J. Y., and Kuo, C. C. (2012) Pivotal role of ADP-ribosylation factor 6 in Toll-like receptor 9-mediated immune signaling. J Biol Chem 287, 4323-4334
68. Peyroche, A., Antonny, B., Robineau, S., Acker, J., Cherfils, J., and Jackson, C. L. (1999) Brefeldin A acts to stabilize an abortive ARF-GDP-Sec7 domain protein complex: involvement of specific residues of the Sec7 domain. Molecular cell 3, 275-285
69. Presley, J. F., Ward, T. H., Pfeifer, A. C., Siggia, E. D., Phair, R. D., and Lippincott-Schwartz, J. (2002) Dissection of COPI and Arf1 dynamics in vivo and role in Golgi membrane transport. Nature 417, 187-193
70. Sciaky, N., Presley, J., Smith, C., Zaal, K. J., Cole, N., Moreira, J. E., Terasaki, M., Siggia, E., and Lippincott-Schwartz, J. (1997) Golgi tubule traffic and the effects of brefeldin A visualized in living cells. The Journal of cell biology 139, 1137-1155
71. Cox, R., Mason-Gamer, R. J., Jackson, C. L., and Segev, N. (2004) Phylogenetic analysis of Sec7-domain-containing Arf nucleotide exchangers. Molecular biology of the cell 15, 1487-1505
72. Mouratou, B., Biou, V., Joubert, A., Cohen, J., Shields, D. J., Geldner, N., Jurgens, G., Melancon, P., and Cherfils, J. (2005) The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf. BMC genomics 6, 20