臺灣博碩士論文加值系統

先天性免疫反應(innate immune responses)，在宿主上建立了第一道防線且會連結並調控後天免疫反應(adaptor immune responses)的啟動來抵抗外來病源菌的入侵；近年來研究的發展中，發現了可以偵測各種入侵體內病源菌的 pattern recognition receptors (PRRs)如 Toll-like receptors (TLRs)和 RIG-I-like receptors (RLRs)，以及探討了 PRRs下游不同訊息調控分子的角色，然而，由於先天性免疫訊息傳遞網複雜度極高，到目前為止還是沒有完全的解開。在實驗室先前的研究中，我們發現了一個新的 adaptor分子，命名為 TAPE (TBK1-Associated Protein in the Endo-lysosomal compartments)，TAPE分布於細胞的 endosome和 lysosome上，且TAPE可以分別活化 IFN-β及 NF-κB pathway。本篇論文研究目的就是要進一步釐清 TAPE如何參與先天免疫的訊息傳遞，以及了解 TAPE在抵抗病毒入侵時扮演了何種的角色。首先我證明 TAPE是位於 TBK1以及 IKKβ上游來分別調控 IFN-β及 NF-κB pathway的活化；此外，過度表現 TAPE也會促使 MAPK家族成員 ERK的磷酸化，磷酸化的 ERK在過去的研究中知道是可以促使細胞生長以及細胞激素(cytokine)的產生。另外在 TAPE-knockdown的細胞株中，發現 TRIF所活化的 IFN-β及 NF-κB pathway明顯的被抑制，但是 IPS-1和 MyD88活化的訊號不會受到 TAPE-knockdown的影響仍然存在；利用 siRNA的方式來進一步研究，證實了 TAPE對於在 endosome的 TLR3和TLR4，以及細胞質中的 MDA5和 RIG-I來活化下游訊號是必需的，但是 TLR5的訊號則不需要 TAPE來傳遞。因此，這些結果證實了TAPE會同時參與位於 endosome以及細胞質中的 PRRs的訊息傳遞，並活化下游並產生對抗病源菌入侵的免疫反應。在未來的研究中，我們會更進一步利用老鼠模式來探討在不同感染疾病中，是否 TAPE也參與其中且會扮演重要角色。

Innate immunity functions to serve the first line of host defense and then links to the initiation and programming of adaptive immunity to build up the full spectrum of immune defenses. Signaling networks underlying the innate immune system have emerged from identification and characterization of various pattern-recognition receptors (PRRs), such as Toll-like receptors (TLRs) and RIG-I-like receptors (RLRs), and innate immune regulators. However, the complexity of these innate immune signaling networks is far from clear yet. Recently our group has identified a novel innate immune adaptor, termed TAPE (TBK1-Associated Protein in the Endo-lysosomal compartments), which is located in the endo-lysosomal compartments and activates the interferon-β (IFN-β) and NF-κB signaling pathways. The objective of my study is to elucidate the TAPE-mediated innate immune signaling pathway and determine the function of TAPE in the anti-viral innate immune responses. My results first revealed that TAPE activated the IFN-β and NF-κB signaling pathways through downstream kinases TBK1 and IKKβ, respectively. Ectopic expression of TAPE also led to the activation of ERK, which is a key protein kinase implicated in cell growth and cytokine production. Using the small interfering RNA approach, I demonstrated that TAPE knockdown impaired the TRIF signal to the IFN-β and NF-κB activation. Furthermore, TAPE knockdown also impaired the TRIF-mediated TLR3 and TLR4 signaling pathways but not the MyD88-mediated TLR5 signaling pathway. Surprisingly, my results also showed that TAPE knockdown could block the RIG-I and MDA-5 signals to the IFN-β activation but not the IPS-1 signal, suggesting that TAPE functions between RLRs and IPS-1. Besides, I further found that TAPE was required for TLR3- and RIG-I-mediated protection against HSV and VSV, respectively. Overall, these results indicate that TAPE is a novel innate immune regulator essential for relaying signals from both endosomal TLRs (TLR3 and TLR4) and cytosolic RLRs to the program of innate immune defenses. Future directions will attempt to determine the implication of TAPE in various infectious diseases in vivo.

1. Introduction .....................................................7
1.1. Innate immune responses and PRRs ..............7
1.1.1. Toll like receptors (TLRs) ...................8

1.2. Innate signal transduction ...........................9
1.2.1. TLR adaptor molecules ........................9
1.2.2. The MyD88-dependent pathway ...............9
1.2.3. The TRIF-dependent pathway .................10
1.2.4. RIG-I-like receptors (RLRs) ..................11
1.2.5. Signal of cytosolic PRRs .................................11
1.2.6. TBK1 and IKKi link TLR and RLR signals to type I IFN induction .12

1.3. Viral recognition in innate immunity ...............................13
1.3.1. PRRs that detect RNA virus ..............................13
1.3.2. PRRs that detect DNA virus ............................14

1.4. Identification of TAPE ....................................15

2. Materials and methods ..........................................17
2.1. Cell lines, cell culture and TLR ligands ........................17
2.2. Plasmids ...........................................17
2.3. Extraction of Plasmid DNA ...................................18
2.4. RNA interference .................................................18
2.5. Transfection ........................................................19
2.6. Western blot analysis and antibodies ...............................20
2.7. Co-immunoprecipitation ...............................21
2.8. Reporter assay ......................................................21
2.9. Enzyme-linked immunosorbent assay (ELISA) ............22
2.10. Plaque assay ...................................................22

3. Results ............................................................................24
3.1. TAPE activates IFN-β through TBK1 ..................................24
3.2. TAPE activates NF-κB pathway through IKKβ and the ERK pathway ...24
3.3. TAPE functions downstream of the TRIF adaptor ..................25
3.4. TAPE is required for the TLR3 and TLR4 signaling pathway .......26
3.5. TAPE is required for the RIG-I and MDA5 signaling pathway .....27
3.6. The NFκB pathway triggered by TLR3, MDA5, or RIG-I would not be blocked in the TAPE-knockdown stable lines...............................................28
3.7. TAPE plays a critical role in the TLR3 and RIG-I mediated anti-viral responses..................................................28

4. Discussion .....................................................29

5. References ..............................................33

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