敗血症主要是由細菌感染引起全身性發炎反應，導致組織受損、器官衰竭、甚至死亡。克雷伯氏肺炎菌（Klebsiella pneumoniae）是導致敗血症的格蘭氏陰性桿菌，具多重抗藥性的克雷伯氏肺炎菌感染導致的爆發已時有報導。巨噬細胞對病原的清除扮演相當重要的角色，但過多的發炎因子反而會造成組織受損。腫瘤壞死因子相關凋亡誘導配體 （tumor necrosis factor-related apoptosis-inducing ligand, TRAIL）除了能夠誘導腫瘤細胞凋亡外，也能夠調控免疫功能。先前研究證實TRAIL能夠增加敗血症小鼠的存活，但TRAIL仍有不穩定的缺點。近年來多種奈米粒子被當作載體應用在藥物輸送系統。由聚氨基酸 （copolypeptide）形成的奈米膠（nanogel）具有高生物相容性，且能減緩藥物或蛋白的半衰期。本研究擬探討TRAIL蛋白經過聚氨基酸奈米膠的包覆後，是否具有更好的治療敗血症效果，共聚氨基酸的組成為60個lysine加上15個threonine (PLL60-b-PLT15)。我們發現奈米膠能夠結合到克雷伯氏肺炎菌上，並且抑制其生長。奈米膠對小鼠肺上皮細胞以及巨噬細胞的毒性在給予脂多醣體刺激後會上升，而將奈米膠直接注射到小鼠肺臟並不會造成小鼠死亡。TRAIL經過nanogel包覆後，增強了小鼠巨噬細胞凋亡訊號，此作用和caspase 3有相關性。TRAIL-nanogel使小鼠巨噬細胞粒線體膜電位受到破壞，且annexin V的量增加，顯示細胞凋亡情況較嚴重。在脂多醣體誘導的小鼠敗血症模式中，TRAIL-nanogel治療後，小鼠血清中腫瘤壞死因子-α （TNF-α）及介白素-6 （interleukin-6）表現量下降，發炎細胞浸潤降低，且腎功能也較良好。而在K. pneumoniae誘導的小鼠敗血症模式中，TRAIL-nanogel治療後的小鼠存活情況較佳，組織發炎較輕微，肺臟中巨噬細胞的數量和肺灌洗液中腫瘤壞死因子-α 及介白素-6的表現量也都降低。整體而言，TRAIL經聚氨基酸奈米膠包覆後，能更加減輕發炎情況，減緩小鼠敗血症造成的組織傷害及死亡。

Sepsis is a systemic inflammatory syndrome caused by bacterial infection, leading to tissue damage, organ failure, and even death. Klebsiella pneumoniae, a gram-negative bacillus, can cause sepsis. Outbreaks of infection due to multidrug-resistant K. pneumoniae have been well documented. Macrophages play an important role in the clearance of pathogens, but excessive inflammatory responses also lead to tissue injury. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) not only induces tumor cell apoptosis, but also regulates immune responses. Previous studies have shown that TRAIL can enhance the survival of septic mice. However, its therapeutic use is hampered due to instability. In recent years, various nanoparticles have been used as carriers in drug delivery systems. Copolypeptide-based nanogel has high biocompatibility and can increase the half-life of drugs or proteins. The aim of this study was to investigate whether TRAIL-conjugated copolypeptide nanogel enhances the therapeutic effect on sepsis. The poly(L-lysine)-block-poly(L-threonine) (PLL-b-PLT) nanogel was composed of 60 lysine residues and 15 threonine residues. We found that the nanogel could bind to K. pneumoniae and inhibit its growth. Toxicity of the nanogel to mouse lung epithelial cells and macrophages was increased after stimulation with lipopolysaccharide (LPS). However, direct injection of the nanogel to lungs did not cause death in mice. Cell apoptotic signal was enhanced by TRAIL-conjugated nanogel in mouse macrophages, which was caspase 3-dependent. TRAIL-nanogel induced the loss of mitochondrial membrane potential and increased the amount of annexin V in mouse macrophages, suggesting that more cells underwent apoptosis after treatment with TRAIL-nanogel. In the LPS-induced sepsis model, treatment with TRAIL-nanogel decreased the expression of TNF-α and interleukin-6, reduced polymorphonuclear cell infiltration, and improved renal functions. In the K. pneumoniae-induced sepsis model, treatment with TRAIL-nanogel prolonged the survival time, reduced tissue inflammation, lowered the numbers of macrophages in the lungs, and reduced the expression of proinflammatory cytokines in the bronchoalveolar lavage fluid (BALF). In conclusion, TRAIL-conjugated copolypeptide nanogel can reduce inflammation and ameliorate sepsis-induced tissue injury and mortality in mice.

Ashkenazi, A., and Dixit, V.M. (1999). Apoptosis control by death and decoy receptors. Curr Opin Cell Biol 11, 255-260.
Beyer, K., Poetschke, C., Partecke, L.I., von Bernstorff, W., Maier, S., Broeker, B.M., and Heidecke, C.D. (2014). TRAIL induces neutrophil apoptosis and dampens sepsis-induced organ injury in murine colon ascendens stent peritonitis. PLoS One 9, e97451.
Beyer, K., Stollhof, L., Poetschke, C., von Bernstorff, W., Partecke, L.I., Diedrich, S., Maier, S., Broker, B.M., and Heidecke, C.D. (2016). TNF-related apoptosis-inducing ligand deficiency enhances survival in murine colon ascendens stent peritonitis. J Inflamm Res 9, 103-113.
Broug-Holub, E., Toews, G.B., van Iwaarden, J.F., Strieter, R.M., Kunkel, S.L., Paine, R., 3rd, and Standiford, T.J. (1997). Alveolar macrophages are required for protective pulmonary defenses in murine Klebsiella pneumonia: elimination of alveolar macrophages increases neutrophil recruitment but decreases bacterial clearance and survival. Infect Immun 65, 1139-1146.
Chen, Y.F., Shiau, A.L., Chang, S.J., Fan, N.S., Wang, C.T., Wu, C.L., and Jan, J.S. (2017). One-dimensional poly(L-lysine)-block-poly(L-threonine) assemblies exhibit potent anticancer activity by enhancing membranolysis. Acta Biomater 55, 283-295.
Condotta, S.A., Cabrera-Perez, J., Badovinac, V.P., and Griffith, T.S. (2013). T-cell-mediated immunity and the role of TRAIL in sepsis-induced immunosuppression. Crit Rev Immunol 33, 23-40.
Cretney, E., McQualter, J.L., Kayagaki, N., Yagita, H., Bernard, C.C., Grewal, I.S., Ashkenazi, A., and Smyth, M.J. (2005). TNF-related apoptosis-inducing ligand (TRAIL)/Apo2L suppresses experimental autoimmune encephalomyelitis in mice. Immunol Cell Biol 83, 511-519.
Crowder, R.N., and El-Deiry, W.S. (2012). Caspase-8 regulation of TRAIL-mediated cell death. Exp Oncol 34, 160-164.
Cziupka, K., Busemann, A., Partecke, L.I., Potschke, C., Rath, M., Traeger, T., Koerner, P., von Bernstorff, W., Kessler, W., Diedrich, S., et al. (2010). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) improves the innate immune response and enhances survival in murine polymicrobial sepsis. Crit Care Med 38, 2169-2174.
Daly, W.H., and Poche, D. (1988). The Preparation of N-Carboxyanhydrides of Alpha-Amino-Acids Using Bis(Trichloromethyl)Carbonate. Tetrahedron Letters 29, 5859-5862.
Dubois, A.V., Midoux, P., Gras, D., Si-Tahar, M., Brea, D., Attucci, S., Khelloufi, M.K., Ramphal, R., Diot, P., Gauthier, F., and Herve, V. (2013). Poly-L-Lysine compacts DNA, kills bacteria, and improves protease inhibition in cystic fibrosis sputum. Am J Respir Crit Care Med 188, 703-709.
Fleischmann, C., Scherag, A., Adhikari, N.K., Hartog, C.S., Tsaganos, T., Schlattmann, P., Angus, D.C., Reinhart, K., and International Forum of Acute Care, T. (2016). Assessment of Global Incidence and Mortality of Hospital-treated Sepsis. Current Estimates and Limitations. Am J Respir Crit Care Med 193, 259-272.
Gaieski, D.F., Edwards, J.M., Kallan, M.J., and Carr, B.G. (2013). Benchmarking the incidence and mortality of severe sepsis in the United States. Crit Care Med 41, 1167-1174.
Giri, K., Ghosh, U., Bhattacharyya, N.P., and Basak, S. (2003). Caspase 8 mediated apoptotic cell death induced by beta-sheet forming polyalanine peptides. FEBS Lett 555, 380-384.
Gurung, P., Rai, D., Condotta, S.A., Babcock, J.C., Badovinac, V.P., and Griffith, T.S. (2011). Immune unresponsiveness to secondary heterologous bacterial infection after sepsis induction is TRAIL dependent. J Immunol 187, 2148-2154.
Gyurkovska, V., and Ivanovska, N. (2016). Distinct roles of TNF-related apoptosis-inducing ligand (TRAIL) in viral and bacterial infections: from pathogenesis to pathogen clearance. Inflamm Res 65, 427-437.
Hickman-Davis, J.M., O'Reilly, P., Davis, I.C., Peti-Peterdi, J., Davis, G., Young, K.R., Devlin, R.B., and Matalon, S. (2002). Killing of Klebsiella pneumoniae by human alveolar macrophages. Am J Physiol Lung Cell Mol Physiol 282, L944-956.
Hoffmann, O., Priller, J., Prozorovski, T., Schulze-Topphoff, U., Baeva, N., Lunemann, J.D., Aktas, O., Mahrhofer, C., Stricker, S., Zipp, F., and Weber, J.R. (2007). TRAIL limits excessive host immune responses in bacterial meningitis. J Clin Invest 117, 2004-2013.
Kelley, S.K., Harris, L.A., Xie, D., Deforge, L., Totpal, K., Bussiere, J., and Fox, J.A. (2001). Preclinical studies to predict the disposition of Apo2L/tumor necrosis factor-related apoptosis-inducing ligand in humans: characterization of in vivo efficacy, pharmacokinetics, and safety. J Pharmacol Exp Ther 299, 31-38.
Kim, T.H., Jiang, H.H., Youn, Y.S., Park, C.W., Lim, S.M., Jin, C.H., Tak, K.K., Lee, H.S., and Lee, K.C. (2011). Preparation and characterization of Apo2L/TNF-related apoptosis-inducing ligand-loaded human serum albumin nanoparticles with improved stability and tumor distribution. J Pharm Sci 100, 482-491.
Kimberley, F.C., and Screaton, G.R. (2004). Following a TRAIL: update on a ligand and its five receptors. Cell Res 14, 359-372.
Kischkel, F.C., Lawrence, D.A., Chuntharapai, A., Schow, P., Kim, K.J., and Ashkenazi, A. (2000). Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5. Immunity 12, 611-620.
Kubota, S., and Fasman, G.D. (1975). The beta conformation of polypeptides of valine, isoleucine, and threonine in solution and solid-state: optical and infrared studies. Biopolymers 14, 605-631.
Lamhamedi-Cherradi, S.E., Zheng, S., Tisch, R.M., and Chen, Y.H. (2003a). Critical roles of tumor necrosis factor-related apoptosis-inducing ligand in type 1 diabetes. Diabetes 52, 2274-2278.
Lamhamedi-Cherradi, S.E., Zheng, S.J., Maguschak, K.A., Peschon, J., and Chen, Y.H. (2003b). Defective thymocyte apoptosis and accelerated autoimmune diseases in TRAIL-/- mice. Nat Immunol 4, 255-260.
LeBlanc, H.N., and Ashkenazi, A. (2003). Apo2L/TRAIL and its death and decoy receptors. Cell Death Differ 10, 66-75.
Liguori, M., Buracchi, C., Pasqualini, F., Bergomas, F., Pesce, S., Sironi, M., Grizzi, F., Mantovani, A., Belgiovine, C., and Allavena, P. (2016). Functional TRAIL receptors in monocytes and tumor-associated macrophages: A possible targeting pathway in the tumor microenvironment. Oncotarget 7, 41662-41676.
Lum, J.J., Pilon, A.A., Sanchez-Dardon, J., Phenix, B.N., Kim, J.E., Mihowich, J., Jamison, K., Hawley-Foss, N., Lynch, D.H., and Badley, A.D. (2001). Induction of cell death in human immunodeficiency virus-infected macrophages and resting memory CD4 T cells by TRAIL/Apo2l. J Virol 75, 11128-11136.
MacFarlane, M., Ahmad, M., Srinivasula, S.M., Fernandes-Alnemri, T., Cohen, G.M., and Alnemri, E.S. (1997). Identification and molecular cloning of two novel receptors for the cytotoxic ligand TRAIL. J Biol Chem 272, 25417-25420.
Mandell, L.A., Wunderink, R.G., Anzueto, A., Bartlett, J.G., Campbell, G.D., Dean, N.C., Dowell, S.F., File, T.M., Jr., Musher, D.M., Niederman, M.S., et al. (2007). Infectious Diseases Society of America/American Thoracic Society consensus guidelines on the management of community-acquired pneumonia in adults. Clin Infect Dis 44 Suppl 2, S27-72.
Muzzarelli, R.A.A. (2009). Genipin-crosslinked chitosan hydrogels as biomedical and pharmaceutical aids. Carbohydrate Polymers 77, 1-9.
Paczosa, M.K., and Mecsas, J. (2016). Klebsiella pneumoniae: Going on the Offense with a Strong Defense. Microbiol Mol Biol Rev 80, 629-661.
Pan, G., Ni, J., Wei, Y.F., Yu, G., Gentz, R., and Dixit, V.M. (1997a). An antagonist decoy receptor and a death domain-containing receptor for TRAIL. Science 277, 815-818.
Pan, G., O'Rourke, K., Chinnaiyan, A.M., Gentz, R., Ebner, R., Ni, J., and Dixit, V.M. (1997b). The receptor for the cytotoxic ligand TRAIL. Science 276, 111-113.
Pitti, R.M., Marsters, S.A., Ruppert, S., Donahue, C.J., Moore, A., and Ashkenazi, A. (1996). Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem 271, 12687-12690.
Reddy, N., Reddy, R., and Jiang, Q. (2015). Crosslinking biopolymers for biomedical applications. Trends Biotechnol 33, 362-369.
Shepard, B.D., and Badley, A.D. (2009). The Biology of TRAIL and the Role of TRAIL-Based Therapeutics in Infectious Diseases. Antiinfect Agents Med Chem 8, 87-101.
Sheridan, J.P., Marsters, S.A., Pitti, R.M., Gurney, A., Skubatch, M., Baldwin, D., Ramakrishnan, L., Gray, C.L., Baker, K., Wood, W.I., et al. (1997). Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors. Science 277, 818-821.
Shima, S., Matsuoka, H., Iwamoto, T., and Sakai, H. (1984). Antimicrobial action of epsilon-poly-L-lysine. J Antibiot (Tokyo) 37, 1449-1455.
Singer, M., Deutschman, C.S., Seymour, C.W., Shankar-Hari, M., Annane, D., Bauer, M., Bellomo, R., Bernard, G.R., Chiche, J.D., Coopersmith, C.M., et al. (2016). The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). JAMA 315, 801-810.
Spierings, D.C., de Vries, E.G., Vellenga, E., van den Heuvel, F.A., Koornstra, J.J., Wesseling, J., Hollema, H., and de Jong, S. (2004). Tissue distribution of the death ligand TRAIL and its receptors. J Histochem Cytochem 52, 821-831.
Steinwede, K., Henken, S., Bohling, J., Maus, R., Ueberberg, B., Brumshagen, C., Brincks, E.L., Griffith, T.S., Welte, T., and Maus, U.A. (2012). TNF-related apoptosis-inducing ligand (TRAIL) exerts therapeutic efficacy for the treatment of pneumococcal pneumonia in mice. J Exp Med 209, 1937-1952.
Stuckey, D.W., and Shah, K. (2013). TRAIL on trial: preclinical advances in cancer therapy. Trends Mol Med 19, 685-694.
Sultana, F., Imran-Ul-Haque, M., Arafat, M., and Sharmin, S. (2013). An Overview of Nanogel Drug Delivery System.
Symonds, P., Murray, J.C., Hunter, A.C., Debska, G., Szewczyk, A., and Moghimi, S.M. (2005). Low and high molecular weight poly(L-lysine)s/poly(L-lysine)-DNA complexes initiate mitochondrial-mediated apoptosis differently. FEBS Lett 579, 6191-6198.
Tian, Y., Tao, T., Zhu, J., Zou, Y., Wang, J., Li, J., Bo, L., and Deng, X. (2013). Soluble tumor necrosis factor related apoptosis inducing ligand level as a predictor of severity of sepsis and the risk of mortality in septic patients. PLoS One 8, e82204.
Turkevich, J., Stevenson, P.C., and Hillier, J. (1951). A study of the nucleation and growth processes in the synthesis of colloidal gold. Discussions of the Faraday Society 11, 55-75.
Vinogradov, S.V. (2010). Nanogels in the race for drug delivery. Nanomedicine (Lond) 5, 165-168.
Walczak, H., Degli-Esposti, M.A., Johnson, R.S., Smolak, P.J., Waugh, J.Y., Boiani, N., Timour, M.S., Gerhart, M.J., Schooley, K.A., Smith, C.A., et al. (1997). TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL. EMBO J 16, 5386-5397.
Warat, M., Sadowski, T., Szliszka, E., Krol, W., and Czuba, Z.P. (2015). The role of selected flavonols in tumor necrosis factor-related apoptosis-inducing ligand receptor-1 (TRAIL-R1) expression on activated RAW 264.7 macrophages. Molecules 20, 900-912.
Wiley, S.R., Schooley, K., Smolak, P.J., Din, W.S., Huang, C.P., Nicholl, J.K., Sutherland, G.R., Smith, T.D., Rauch, C., Smith, C.A., and et al. (1995). Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity 3, 673-682.
Yadav, H.K., Al Halabi, N.A., and Alsalloum, G.A. (2017). Nanogels as Novel Drug Delivery Systems-A Review. Insights in Pharma Research 1.
Yao, Z., Zhang, P., Guo, H., Shi, J., Liu, S., Liu, Y., and Zheng, D. (2015). RIP1 modulates death receptor mediated apoptosis and autophagy in macrophages. Mol Oncol 9, 806-817.
Yeaman, M.R., and Yount, N.Y. (2003). Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55, 27-55.
Zhu, D.M., Shi, J., Liu, S., Liu, Y., and Zheng, D. (2011). HIV infection enhances TRAIL-induced cell death in macrophage by down-regulating decoy receptor expression and generation of reactive oxygen species. PLoS One 6, e18291.