- 學位論文
E3泛素連接酶ZNRF1在TLR3誘發之免疫反應中的角色
The Role of the E3 Ubiquitin Ligase ZNRF1 in TLR3-driven Immune Response
摘要
類鐸受體(Toll-like receptors)在先天性免疫反應中扮演著重要的角色。這類型的受體藉由辨識病原體上特定的構造(pathogen-associated molecular patterns, PAMPs)以及從壞死或受到刺激細胞所釋放出來的物質(damage-associated molecular patterns, DAMPs)達到宿主對抗外來病原體的入侵。我們先前嘗試去尋找調控第四類鐸受體(TLR4)的調控因子，並發現Znrf1基因在巨噬細胞受到第四類鐸受體的受質脂多醣(lipopolysaccharide, LPS)的刺激後會被誘發起來。ZNRF1在先前的研究中已被報導和神經細胞變性(neural degeneration)有關，然而其在先天性免疫中所扮演的角色仍是未知的。我們先前的研究結果證實ZNRF1透過調控微囊(caveolae)構造中的微囊蛋白1 (Caveolin-1)的降解，促進前發炎激素(pro-inflammatory cytokines)的製造，並抑制介白素10 (Interleukin-10, IL-10)這個抗發炎激素的產生，此顯示著ZNRF1在第四類鐸受體中扮演著正調控的角色。然而其在核內體上的類鐸受體(endosomal TLRs)所誘發的免疫反應中扮演的角色仍是未知的。因此本研究的主旨為探討其在這類型受體，特別是針對第三類鐸受體(TLR3)，所調控的免疫反應中扮演的角色進行探討。我們的研究結果顯示在巨噬細胞中，若第三、第七、第九類鐸受體活化後，Znrf1基因的剔除會促進前發炎激素和第一型干擾素(type I interferons)的製造。我們更進一步尋找可能的調控機轉。我們發現在第三類鐸受體活化後，ZNRF1會透過和受體結合促使其被送往溶酶體(lysosome)降解。此外，我們也發現微囊蛋白1參與在此調控機轉中。在巨噬細胞中，Znrf1基因剔除所導致微囊蛋白1表現量的提升會去抑制溶酶體的功能。總結來說，本研究發現ZNRF1利用調控第三類鐸受體降解，負調控其所引發的免疫反應。
並列摘要
Toll-like receptors (TLRs) play crucial roles in innate immune response against pathogenic infection by recognition of specific pathogen-associated molecular patterns (PAMPs) or endogenous damage-associated molecular patterns (DAMPs) released from damaged or stressed cells. We previously sought to identify regulators involved in the regulation of TLR4-mediated immune response, and found that ZNRF1, which was induced upon TLR4 activation by LPS challenge in macrophages. ZNRF1 was reported to be involved in neural degeneration, but its role in innate immune response remains unclear. Our previous studies demonstrated that ZRNF1 enhanced the production of pro-inflammatory cytokines and suppressed anti-inflammatory cytokine IL-10 expression upon LPS stimulation through controlling the protein stability of CAV1, a crucial component of caveolae. It suggests that ZNRF1 positively regulates TLR4-triggered inflammatory response. However, its role in endosomal TLRs (TLR3/7/9)-driven immune responses remains unclear. Thus in this study, we aim to characterize its function in inflammatory responses triggered by endosomal TLRs, especially focusing on TLR3. Our results show that the production of pro-inflammatory cytokines and type I interferons is significantly increased in Znrf1-depleted macrophages after activation of three endosomal TLRs. We further explore the possible regulatory mechanism and find that ZNRF1 associates with TLR3 and controls protein stability of TLR3 through the lysosome-mediated degradation after receptor activation. In addition, we demonstrate that CAV1 is involved in this regulation. Elevated CAV1 protein level caused by ZNRF1 depletion suppresses lysosomal function in macrophages upon TLR3 engagement. Taken together, this study suggests that ZRNF1 negatively regulates TLR3-mediated immune response through the control of receptor stability.