呼吸器相關性肺炎是加護病房中患者最常見的院內感染，綠膿桿菌在加護病房外極少引起肺炎，但卻是引發呼吸器相關性肺炎最常見的致病菌，進而造成嚴重的感染，其原因目前仍不甚清楚。因此，為探討綠膿桿菌如何增加呼吸器相關性肺炎的發生，WT和JNK1-/-小鼠先由鼻腔注入綠膿桿菌並於兩天後連接呼吸器三小時再進行肺部損傷的分析。另使用分離自WT、JNK1-/-和IKK△mye小鼠的巨噬細胞進行綠膿桿菌的體外刺激後收集上清液檢測發炎性細胞激素的產生，並分別由WT和JNK1-/-小鼠的鼻腔給予取自WT小鼠的巨噬細胞上清液，一小時後連接呼吸器三小時再分析肺部的損傷。結果顯示，先給予綠膿桿菌或上清液後再連接呼吸器時，WT小鼠肺部的嗜中性白血球浸潤程度、肺泡沖提液的發炎性細胞激素和一氧化氮的含量皆明顯地增加，而巨噬細胞經體外刺激後收集的上清液中顯著增加的TNF-α極可能在呼吸器相關性肺炎的發生扮演重要的角色。當給予綠膿桿菌後再連接呼吸器時，JNK1-/-小鼠的肺部發炎及損傷均減緩，但JNK1-/-小鼠的巨噬細胞受刺激後，上清液中TNF-α的含量仍有明顯的增加。然而當IKK△mye小鼠的巨噬細胞受刺激後，上清液中TNF-α的含量則明顯下降。綜合以上結果，本研究模式中，綠膿桿菌經由活化巨噬細胞的NF-κB增加TNF-α的生成，活化肺部組織內JNK的訊息傳遞路徑，而增加小鼠呼吸器相關性肺炎的發生。

Ventilator-associated pneumonia (VAP) is a common nosocomial infection among intensive care unit (ICU) patients and the most common frequent causative microorganism is P. aeruginosa although it rarely causes pneumonia outside the ICU. To study the pathogenesis mechanism of VAP caused by P. aeruginosa colonization, first, C57BL/6 (WT) mice and JNK1 knockout (JNK1-/-) mice received ventilation for 3 hr at 2 days after receiving nasal instillation of P. aeruginosa to induce lung injury. Second, alveolar macrophages (AMs) isolated from WT, JNK1-/- and IKK△mye (deletion of IκB kinase in myeloid cells) mice were ex vivo stimulated with live P. aeruginosa and supernatants were collected for proinflammatory cytokine assay. WT and JNK1-/- mice also received ventilation at 1 hr after receiving nasal instillation of the collected supernatant to induce lung injury. Instillation of P. aeruginosa before ventilation significantly increased the neutrophil sequestration in lungs as well as the levels of pro-inflammatory cytokines and nitrite in bronchoalveolar lavage fluid of WT mice. Instillation of supernatant before ventilation induced more severe lung injury in WT mice and the proinflammatory cytokine assay of supernatant indicated that TNF-α is a critical regulator of VAP induced by P. aeruginosa. Moreover, AMs of IKK△mye mice showed decreased production of TNF-α after ex vivo stimulation but not the AMs of JNK1-/- mice, and instillation of P. aeruginosa before ventilation induced less lung injury in JNK1-/- mice. These results suggest that the pathogenesis mechanism of VAP caused by P. aeruginosa involves production of TNF-α through activation of NF-κB in alveolar macrophages and JNK signaling pathway in lung tissue.

Acknowledgement ... ... ... ... ...i
Abstract in Chinese ... ... ... ... ...ii
Abstract in English ... ... ... ... ...iii
Introduction ... ... ... ... ... ... ... ... ...1
Materials and Methods ... ... ... ...8
Results ... ... ... ... ... ... ... ... ... ... ...17
Discussion ... ... ... ... ... ... ... ... ...24
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Appendix ... ... ... ... ... ... ... ... ... ...56
References ... ... ... ... ... ... ... ... ...57

1. Webb HH, Tierney DF. Experimental pulmonary edema due to intermittent positive pressure ventilation with high inflation pressures. 1974. Am Rev Respir Dis 110:556-65.
2. Heyland DK, Cook DJ, Griffith L, Keenan SP, Brun-Buisson C. 1999.The attributable morbidity and mortality of ventilator-associated pneumonia in the critically ill patient. Am J Respir Crit Care Med 159(4 Pt 1):1249-56.
3. Budweiser S, Jorres RA, Pfeifer M. 2008. Treatment of respiratory failure in
COPD. Int J Chron Obstruct Pulmon Dis 3:605-18
4. Halbertsma FJ, Vaneker M, Scheffer GJ, van der Hoeven JG. Cytokines and biotrauma in ventilator-induced lung injury: a critical review of the literature. 2005. Neth J Med 63:382-92
5. Dos Santos CC, Slutsky AS. 2000. Invited review: mechanisms of ventilator-induced lung injury: a perspective. J Appl Physiol 89:1645-55.
6. Bowden DH. The Alveolar Macrophage. 1984. Environ Health Perspect 55:327-41.
7. Valencia M, Torres A. Ventilator-associated Pneumonia. 2009. Curr Opin Crit Care 15:30-5.
8. Ader F, Jawhara S, Nseir S, Kipnis E, Faure K, Vuotto F, Chemani C, Sendid B, Poulain D, Guery B. Short term Candida albicans colonization reduces Pseudomonas aeruginosa-related lung injury and bacterial burden in a murine model. 2011. Crit Care 15:R150.
9. Steven M. Koenig and Jonathon D. Truwit. Ventilator-Associated Pneumonia: Diagnosis, Treatment, and Prevention. 2008. Clin Microbiol Rev 19:637-657
10. Crouch Brewer S, Wunderink RG, Jones CB, Leeper KV Jr. Ventilator-Associated Pneumonia Due to Pseudomonas Aeruginosa. 1996. Chest 109:1019-29.
11. Berra L, Sampson J, Wiener-Kronish J. Pseodomonas aeruginosa: acute lung injury or ventilator-associated pneumonia? 2010. Minerva Anestesiol 76:824-32.
12. Lederberg, Joshua et al. Pseudomonas. Encyclopedia of Microbiology. Second Edition. Volume 3. San Diego, 2000. p.876-891.
13. Botzenhardt, K., and Doring, G. Ecology and epidemiology of Pseudomonas aeruginosa. Pseudomonas aeruginosa as an Opportunistic Pathogen. 1993. p.1-7.
14. El Solh AA, Alhajhusain A. Update on the treatment of Pseudomonas aeruginosa pneumonia. 2009. J Antimicrob Chemother 64:229-38.
15. Parker D, Prince A. Innate Immunity in the Respiratory Epithelium. 2011. Am J Respir Cell Mol Biol 45:189-201.
16. Bowden DH. The Alveolar Macrophage. 1984. Environ Health Perspect 55:327-41.
17. Sibille Y, Reynolds HY. Macrophages and polymorphonuclear neutrophils in lung defense and injury. 1990. Am Rev Respir Dis 141:471-501.
18. Spelman K, Burns J, Nichols D, Winters N, Ottersberg S, Tenborg M. Modulation of cytokine expression by traditional medicines: a review of herbal immunomodulators. 2006. Altern Med Rev 11:128-50.
19. Mora AL, Torres-González E, Rojas M, Corredor C, Ritzenthaler J, Xu J, Roman J, Brigham K, Stecenko A. Activation of alveolar macrophages via the alternative pathway in herpesvirus-induced lung fibrosis. 2006. Am J Respir Cell Mol Biol 35:466-73.
20. Lin CY, Zhang H, Cheng KC, Slutsky AS. Mechanical ventilation may increase susceptibility to the development of bacteremia. 2003. Crit Care Med 31(5):1429-34.
21. Mercedes Rincon, Charles G. Irvin. Role of IL-6 in Asthma and Other Inflammatory Pulmonary Diseases. 2012. Int J Biol Sci 8:1281-1290.
22. Paul J. Wolters, Charlie Wray, Rachel E. Sutherland, Sophia S. Kim, Jon Koff, Ying Mao, James A. Frank. Neutrophil-Derived IL-6 Limits Alveolar Barrier Disruption in Experimental Ventilator-Induced Lung Injury. 2009. J Immuno 182:8056-8062.
23. NA Eisele, DM Anderson. Host Defense and the Airway Epithelium: Frontline Responses That Protect against Bacterial Invasion and Pneumonia. 2011. J Pathog 249802
24. Bals R, Hiemstra PS. Innate immunity in the lung: how epithelial cells fight against respiratory pathogens. 2004. Eur Respir J 23:327-33.
25. Bradley JR. TNF-mediated inflammatory disease. 2008. J Pathol 2214:149-60.
26. Bertok S, Wilson MR, Morley PJ, de Wildt R, Bayliffe A, Takata M. Selective inhibition of intra-alveolar p55 TNF receptor attenuates ventilator-induced lung injury. 2012 Thorax 67:244-51.
27. Noda E, Hoshina H, Watanabe H, Kawano T. Production of TNF-α by polymorphonuclear leukocytes during mechanical ventilation in the surfactant-depleted rabbit lung. 2003. Pediatr Pulmonol 36:475-81.
28. Srivastava SK, Ramana KV. Focus on Molecules: Nuclear Factor-kappaB. 2009. Exp Eye Res 88:2-3.
29. Baud V, Karin M. Signal transduction by tumor necrosis factor and its relatives. 2001. Trends Cell Biol 11:372-7.
30. Bode AM, Dong Z. The Functional Contrariety of JNK. 2007. Mol Carcinog 46:591-8.
31. Li LF, Yu L, Quinn DA. Ventilation-induced neutrophil infiltration depends on c-Jun N-terminal kinase. 2004. Am J Respir Crit Care Med. 169:518-24
32. Jia J, Alaoui-El-Azher M, Chow M, Chambers TC, Baker H, Jin S. c-Jun NH2-terminal kinase-mediated signaling is essential for Pseudomonas aeruginosa ExoS-inducedapoptosis. 2003. Infect Immun 71:3361-70.
33. Waetzig V, Herdegen T. Context-specific inhibition of JNKs: overcoming the dilemma of protection and damage. 2005. Trends Pharmacol Sci 26:455-61.
34. Yin Y, Wang S, Sun Y, Matt Y, Colburn NH, Shu Y, Han X. JNK/AP-1 pathway is involved in tumor necrosis factor-a induced expression of vascular endothelial growth factor in MCF7 cells. 2009. Biomed Pharmacother 63:429-35.
35. Hirosumi J, Tuncman G, Chang L, Görgün CZ, Uysal KT, Maeda K, Karin M, Hotamisligil GS. A central role for JNK in obesity and insulin resistance. 2002. Nature 420:333-6.
36. Nguyen MT, Satoh H, Favelyukis S, Babendure JL, Imamura T, Sbodio JI, Zalevsky J, Dahiyat BI, Chi NW, Olefsky JM. JNK AND TNF-a MEDIATE FREE FATTY ACID-INDUCED INSULIN RESISTANCE IN 3T3-L1 ADIPOCYTES. 2005. J Biol Chem 280:35361-71.
37. Fujioka S, Niu J, Schmidt C, Sclabas GM, Peng B, Uwagawa T, Li Z, Evans DB, Abbruzzese JL, Chiao PJ. NF-κB and AP-1 Connection: Mechanism of NF-κB –Dependent Regulation of AP-1 Activity. 2004. Mol Cell Biol 24:7806-19.
38. Carnesecchi S, Deffert C, Pagano A, Garrido-Urbani S, Métrailler-Ruchonnet I, Schäppi M, Donati Y, Matthay MA, Krause KH, Barazzone Argiroffo C. NADPH oxidase-1 plays a crucial role in hyperoxia-induced acute lung injury in mice. 2009. Am J Respir Crit Care Med 180:972-81.
39. Chen LW, Chang WJ, Wang JS, Hsu CM. Interleukin-1 mediates thermal injury-induced lung damage through C-Jun NH2 -terminal kinase signaling. 2007. Crit Care Med 35:1113-22.
40. Hui Su Lee, Hee Jae Kim, Chang Sook Moon, Young Hae Chong, Jihee Lee Kang. Inhibition of c-Jun NH2-terminal kinase or extracellular signal-regulated kinase improves lung injury. 2004. Respir Res 5: 23.
41. Li LF, Liao SK, Ko YS, Lee CH, Quinn DA. Hyperoxia increases ventilator-induced lung injury via mitogen-activated protein kinases: a prospective, controlled animal experiment. 2007. Crit Care 11:R25.
42. Dolinay T, Wu W, Kaminski N, Ifedigbo E, Kaynar AM, Szilasi M, Watkins SC, Ryter SW, Hoetzel A, Choi AM. Mitogen-Activated Protein Kinases Regulate Susceptibility to Ventilator-Induced Lung Injury. 2008. PLoS One 3:e1601.
43. Liu YY, Liao SK, Huang CC, Tsai YH, Quinn DA, Li LF. Role for nuclear factor-kappaB in augmented lung injury because of interaction between hyperoxia and high stretch ventilation. 2009. Transl Res 154:228-40.
44. Miranda KM, Espey MG, Wink DA. A Rapid, Simple Spectrophotometric Method for Simultaneous Detection of Nitrate and Nitrite. 2001. Nitric Oxide 5:62-71.