先天性免疫系統與肝臟壓力反應失調參與於克雷白氏肺炎桿菌引發糖尿病患肝膿瘍之致病過程。

Dysregulation of innate immune system and hepatic stress responses involve in pathogenesis of Klebsiella pneumoniae-induced liver abscess associated with diabetes mellitus.

彭佳琇(Jia-Shiou Peng)

指導教授 ： 詹明修

中山醫學大學/醫學院/免疫學研究所/碩士(2007年)

https://doi.org/10.6834/CSMU.2007.00058

摘要

克雷白氏肺炎桿菌 (Klebsiella pneumoniae) 為格蘭氏陰性腸內桿菌科 (Enterobacteriacea) 的細菌，經常引起社群及院內感染。在台灣，此菌的感染好發於糖尿病患，易併發多種臨床症狀，包括原發性肺炎、化膿性肝膿瘍 (pyogenic liver abscess)、腦膜炎 (meningitis) 以及轉移性眼內炎 (metastatic endophthalmitis) 及其高致死率，故 K. pneumoniae 引發糖尿病患肝膿瘍已成為國內重要的感染症。K. pneumoniae 的致病毒力因子包括莢膜 (capsule)、脂多醣體 (lipopolysaccharide, LPS) 等多種，但何者為主要致病因子仍不清楚。另一方面，糖尿病較易受 K. pneumoniae 感染而引發肝膿瘍之宿主成因亦仍需進一步確認。為研究糖尿病宿主之相關致病因素，我們對糖尿病人或建立糖尿病鼠的先天性免疫力，及肝臟對感染之反應進行研究。實驗發現糖尿病人的嗜中性細胞和巨噬細胞吞噬 K. pneumoniae 的能力相對地比非糖尿病人的低。檢測免疫細胞移行的能力，發現糖尿鼠的免疫細胞移行能力較正常鼠弱，且細胞的死亡率較高。比較 K. pneumoniae 感染小鼠的存活率，糖尿鼠感染 K. pneumoniae 後其死亡率較正常鼠高。另一方面，糖尿鼠經莢膜黏性較低或不具黏性的 K. pneumoniae 菌株感染後，肝細胞間隙有變大的現象，然而莢膜黏性高者感染後，糖尿鼠肝組織則呈現空洞狀，此現象未見於正常鼠肝細胞中。進一步比較小鼠肝臟細胞訊息傳遞路徑活化的差異，發現小鼠灌食感染 K. pneumoniae 後，糖尿鼠肝細胞 caspase 3 活性增加。有趣地，糖尿鼠肝細胞 Erk 及 p38 的活化程度高於正常鼠之肝細胞。而不論是否有 K. pneumoniae 的感染，正常鼠之內質網壓力反應 (unfolded protein responses, UPR) 的程度較高於糖尿鼠，且也發現肝臟有細胞自噬 (autophagy) 發生。以人類肝癌細胞株 Hep G2 及 BALB/c 肝細胞株 (BNL) 觀察 K. pneumoniae 引發細胞凋亡的情形，發現與活菌共同培養下，Hep G2 及 BNL 細胞內 DNA 片段化程度增加，但若以去莢膜的 K. pneumoniae 感染細胞，則不會造成細胞的凋亡，所以 K. pneumoniae 所引起的肝細胞凋亡，莢膜可能扮演了某種角色。綜合以上的結果，我們認為除了 K. pneumoniae 所擁有之特定致病因子外，糖尿病引發免疫低下與肝細胞低應變能力亦是導致 K. pneumoniae 引發肝膿瘍的原因。

關鍵字

糖尿病 ； 克雷白氏肺炎桿菌 ； 肝膿瘍

並列摘要

Klebsiella pneumoniae, an important pathogen belonging to Gram-negative Enterobacteriaceae, usually causes community- and hospital-acquired infections. In Taiwan, diabetic patients have an increased susceptibility to K. pneumoniae infections combined with several clinical syndromes, including primary pneumonia, pyogenic liver abscess, meningitis and metastatic endophthalmitis, and their high mortalities, K. pneumoniae-induced liver abscess in diabetic patients become an important infectious disease in Taiwan. The virulence factors of K. pneumoniae identified include capsule, lipopolysaccharide (LPS) et al., however, the dominant pathogenic factor(s) was (were) still unclear. On the other hand, the host factors involved in pathogenesis of K. pneumoniae-induced liver abscess associated with diabetes mellitus also remain to be clarified. For studying the host effect(s) of diabetes on K. pneumoniae-induced liver abscess, we evaluated the innate immune activities of diabetes mellitus patients or diabetic mice and evaluated the responses of liver tissue to K. pneumoniae infection. Our results indicated the abilities of phagocytosis of neutrophils and macrophages from diabetic patients decreased. The migration capabilities and survival rates of both immune cells from diabetic mice were also less than those obtained from non-diabetic mice. Comparing the survival rates, K. pneumoniae-infected diabetic mice had a higher fatality rate with K. pneumoniae-infection than control non-diabetic mice. After infected with K. pneumoniae with viscoid capsule, the interspace between the hepatocytes of diabetic mice were formed as cleft, however, hyper-mucoid K. pneumoniae caused lots of vacuoles within hepatic tissues in diabetic mice but not non-diabetic groups. Furthermore, we evaluated the activities of intracellular signal pathways in hepatic tissues. After oral administration with K. pneumoniae, caspase 3 activity was detected in liver samples from K. pneumoniae-infected diabetic mice but not other groups. Interestingly, activities of Erk and p38 were more higher in hepatic tissues from diabetic mice than those samples from non-diabetic mice. Whether mice were K. pneumoniae-infected, unfolded protein responses (UPR) in liver samples of non-diabetic mice were more active than in diabetic mouse livers, and the autophagy were also be deteted in diabetic mouse liver tissues. To evaluate the direct cytopathic effects of K. pneumoniae in vitro, HepG2 or BNL cells were cocultured with K. pneumoniae and the apoptosis of these cells were the determined. Our results indicated that the DNA framgmentation increased in a dose dependent manner by live but not dead microorganisms. We proposed that capsule maybe play a role in hepatic cytopathy. Taken together, except specific virulent factors of K. pneumoniae, the immunocompromise and low stress-defenses in diabetes mellitus may also result in K. pneumoniae-induced liver abscess.

並列關鍵字

Dabetes mellitus ； Klebsiella pneumoniae ； Liver abscess

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