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研究生：	徐政倫 Hsu, Cheng-Lun
論文名稱：	探討TAPE-like (TAPE-L) 分子在先天免疫調控及細菌感染之角色 Functional study of TAPE-like (TAPE-L) adaptor in innate immune regulation and bacterial infection
指導教授：	凌斌 Ling, Pin
學位類別：	碩士 Master
系所名稱：	醫學院 - 微生物及免疫學研究所 Department of Microbiology & Immunology
論文出版年：	2014
畢業學年度：	102
語文別：	英文
論文頁數：	53
中文關鍵詞：	TAPE分子、TAPE-L分子、Toll-like receptor (TLR) 、先天性免疫
外文關鍵詞：	TBK1-associated protein in endolysosomes (TAPE), TAPE-like (TAPE-L), Toll-like receptor (TLR), innate immunity
相關次數：	點閱：44 下載：0
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先天免疫系統偵測病原感染主要藉由Pattern recognition receptors (PRRs)，PRR可以辨識外來病原體具高度保留的Pathogen-associated molecular pattern (PAMPs)，其中Toll-like receptors (TLRs) 可以偵測細菌入侵並引起下游的免疫發炎反應，目前已經有相當多的文獻探討TLRs如何去活化先天免疫反應的分子機制。我們實驗室之前的研究已經發現一個新的先天免疫調控分子，TAPE會影響TLR3, TLR4, 及RIG-I-like receptors (RLRs) 訊息傳遞，另外，之前實驗室在人類基因體資料庫發現另一個TAPE旁系同源分子，我們將其命名為TAPE-like (TAPE-L)。先前實驗室初步的生化實驗已證實TAPE-L會活化下游的轉錄活化因子NF-κB，並參與細胞表面上的TLRs訊息傳遞路徑，我將進一步地利用基因剃除 (knockout) 小鼠、活體外細胞及人類單核球細胞株THP-1等材料來佐證TAPE-L在先天免疫調控的角色。實驗結果顯示TAPE-L基因默化 (knockdown) 會阻礙TLRs活化下游的NF-κB及發炎性細胞激素表達，在TAPE-L基因剃除細胞中，也可以發現LPS誘發的IκB降解作用及發炎反應都有明顯缺陷，而TAPE-L 基因剔除小鼠對LPS引起的敗血症的抵抗性則顯示TAPE-L 在TLR4訊息傳遞路徑的重要性。另外，我們也發現TAPE-L 基因剃除小鼠較wild type小鼠容易死於格蘭氏陰性菌感染，顯示TAPE-L 在活體內抵抗細菌感染的重要性，此外在細菌刺激下，TAPE-L 缺乏也能觀察到降低的發炎性細胞激素表達量，顯示TAPE-L會參與細菌引起的發炎反應。總結以上實驗結果，無論是在活體外細胞或活體實驗上，我證實了TAPE-L會參與調控細胞表面TLRs訊息活化路徑，同時也發現TAPE-L對於抗細菌免疫機制的重要性。未來的研究將更進一步地探索TAPE-L在細菌感染中其他可能參與的TLRs和PRRs訊息傳遞路徑。

Innate immune recognition of pathogen infection is mainly mediated by pattern recognition receptors (PRRs) upon sensing the pathogen molecular signatures, termed pathogen-associated molecular patterns (PAMPs). Toll-like receptors (TLRs) represent a prototype family of PRRs to detect a variety of microbial components and trigger innate immune defenses. Much attention and progress have focused on understanding the downstream signaling pathways linking TLRs to innate immune activation. Previous work from our lab uncovered an innate immune regulator, called TAPE (TBK1-associated protein in endolysosome), which is involved in TLR3, TLR4, and RIG-I-like receptor signaling pathways. Furthermore, a TAPE paralog protein termed TAPE-like (TAPE-L) was identified by blast analysis. Our preliminary biochemical analyses showed that TAPE-L was a potent NF-қB activator and capable of regulating the surface TLR pathways. We further used gene knockout mice, primary macrophages and THP-1 cells to assess whether TAPE-L functions as an innate immune regulator. Results showed that gene silencing of TAPE-L impaired the activation of NF-κB and inflammatory cytokine production upon TLR agonist stimulation, indicating that TAPE-L was involved in linking surface TLRs signaling to the activation of NF-κB. Tape-l -/- cells also displayed defective LPS-induced IκB degradation and cytokine responses. The resistance to LPS-induced sepsis in Tape-l -/- mice supports the in vivo role of TAPE-L in TLR4 signaling. Furthermore, Tape-l -/- mice were more susceptible to gram-negative bacterial infection than wild type mice, suggesting the in vivo significance of TAPE-L for antibacterial defenses. TAPE-L-deficient macrophages also showed impaired inflammatory cytokine responses to bacteria, indicating that TAPE-L facilitated the inflammation response against bacterial infection. Collectively, my thesis work demonstrated that TAPE-L is essential for activating the surface TLRs pathway and required for antibacterial immunity. My future directions will aim to explore the potential roles of TAPE-L in regulating other TLR or PRR pathways during bacterial infections.

摘要	I
Abstract	II
誌謝	IV
Table of contents	V
List of figures	VIII
Introduction	1
1 Innate immunity and PRRs	1
2 PRRs and antibacterial immunity	1
3 NOD-like receptors (NLRs) and inflammasome	2
4 Toll-like receptors (TLRs)	2
5 The TLR signaling pathway	3
6 MyD88- and TRIF-dependent TLR pathways	3
7 TBK1 and autophagic clearance of bacteria	4
8 Role of TAPE in innate immune pathways	5
9 Biological role of TAPE-like (TAPE-L)	6
Materials and methods	7
1 Bacterial strains	7
2 Mice	7
3 Cell culture and reagents	8
4 Isolation of primary macrophages	8
5 Lentiviral vectors and shRNA plasmids	9
6 RNA interference	9
7 Animal infection	10
8 Cell culture and infection	11
9 Secreted embryonic alkaline phosphatase (SEAP) assay	11
10 Enzyme-linked immunosorbent assay (ELISA)	12
11 SDS-PAGE and Western blot	12
12 Real time PCR	12
13 Lactose dehydrogenase (LDH) release assay	13
Results	14
1 TAPE-L is involved in the cell surface Toll-like receptor pathways.	14
2 TAPE-L is required to trigger inflammation upon bacterial infection.	14
3 Generation of TAPE-L-deficient mice	15
4 Ex vivo role of TAPE-L in regulating the TLR4-NF-κB pathway	16
5 In vivo role of TAPE-L during Salmonella Typhimurium infection	17
6 In vivo role of TAPE-L during Vibrio vulnificus infection	17
7 In vivo role of TAPE-L during LPS-induced septic shock.	18
Discussion	19
1 Role of TAPE-L in antibacterial immunity	19
2 Role of TAPE-L in the TLR pathways	19
3 The relationship among TAPE-L-mediated immunity and antibacterial defenses	20
4 Potential role of TAPE-L in other PRR pathways	22
5 TAPE-L-mediated immunity might be cell type-dependent.	24
6 Potential role of TAPE-L in cell-autonomous bacterial defenses in macrophages	24
7 Different responsiveness to endotoxic shock among substrains of C57BL/6 mice	25
References	26
Figures and Figure legends	33
Appendix	45
1 Cytokine profile of LPS-stimulated fetal liver macrophages	45
2 TAPE-L-mediated immunity might be cell type-dependent.	47
3 Role of TAPE-L for involving the immune responses against Escherichia coli	48
4 Bacterial burden of Salmonella Typhimurium-infected mice	49
5 Cytokine profile of the gram-negative bacteria-infected mice	51
6 Survival of Tape-l-/-mice upon the lethal Vibrio vulnificus infection	52
7 C57BL/6N mice are more resistant to LPS-induced septic shock than C57BL/6J mice.	53
                                    

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