ZNRF1調控第七/九型類鐸受體誘發的發炎反應之功能

Abstract

第七型類鐸受體(TLR7)及第九型類鐸受體(TLR9)是兩個表現於漿細胞樣樹突細胞(plasmacytoid dendritic cells)的胞內體(endosome)類鐸受體，他們會辨識核酸產生大量的第一型干擾素(type I interferons)來抵禦病毒。然而過多的第一型干擾素可能會造成問題如自體免疫疾病，因此第一型干擾素的產生需要嚴密調控。我們實驗室過去發現一個E3泛素接合酶(E3 ubiquitin ligase) ──ZNRF1會透過促進caveolin-1的泛素化(ubiquitination)及降解(degradation)來正向調控第四型類鐸受體(TLR4)訊息傳遞路徑。然而，在第三型類鐸受體(TLR3)介導的免疫反應中，ZNRF1作為一個負向調控者會對第三型類鐸受體進行泛素化並造成其走向溶酶体式(lysosomal)降解。本篇研究我們發現在R848(第七型類鐸受體的配體)及CpG A(第九型類鐸受體的配體)的刺激下，類FMS酪氨酸激酶-3受體所分化的漿細胞樣樹突細胞(Flt3L-pDCs)或人類漿細胞樣樹突細胞(CAL-1)在ZNRF1缺失的情況下會增加第一型干擾素的表現，而ZNRF1的E3泛素接合酶活性對於其負向調控第七型類鐸受體誘發的第一型干擾素產生是重要的。類似於TLR3，ZNRF1會與TLR7及TLR9互動。ZNRF1會促進在第九型類鐸受體的離胺酸(lysine)932位點(residue)進行的離胺酸63型多泛素化 (K63-linked polyubiquitination)，而這樣的調控是需要其E3泛素接合酶活性。我們更進一步發現ZNRF1缺失的老化老鼠有脾臟腫大、動脈周圍淋巴鞘(periarteriolar lymphoid sheath)擴大及自體抗體增加的現象。去除第七型類鐸受體或骨髓分化因子八八蛋白質(MyD88)後減緩這些表現型(phenotype)，意味著ZNRF1有潛力扮演預防第七型類鐸受體所致自體免疫的角色。總結來說，我們認為ZNRF1負向調控第七及第九型類鐸受體訊息傳遞，並藉由降低過量第一型干擾素的產生以減緩自體免疫疾病的發展。

Toll-like receptor (TLR) 7 and TLR9 are two of the endosomal TLRs that expressed in plasmacytoid dendritic cells (pDCs). They sense nucleic acids and produce indispensable amounts of type I interferons (IFNs) for anti-viral response. However, excessive type I IFNs may lead to several disadvantages such as the autoimmunity, therefore type I IFNs production needs to be tightly regulated. Our lab previously found that one of the E3 ubiquitin ligases — Zinc and RING finger 1 (ZNRF1) positively regulates TLR4 signaling pathway through promoting caveolin-1 ubiquitination and degradation. However, in TLR3-mediated immune response, ZNRF1 serves as a negative regulator by ubiquitinating TLR3 for lysosomal degradation. In this study, we discovered that deletion of ZNRF1 in primary mouse fms-like tyrosine kinase 3 ligand (Flt3L)-driven pDCs or human pDCs, CAL-1, enhances type I IFNs mRNA expression after stimulation of R848 (TLR7 ligand) and CpG A (TLR9 ligand). Besides, ZNRF1 E3 ubiquitin ligase activity is required for its negative regulation of TLR7-driven type I IFN production. Similar to TLR3, ZNRF1 interacts with TLR7 and TLR9, and promotes K63-linked polyubiquitination on TLR9 lysine 932 residue in an E3 ubiquitin ligase activity-dependent manner. We further discovered that aged Znrf1-/- mice showed splenomegaly, enlarged periarteriolar lymphoid sheath and increased autoantibodies production. Depletion of TLR7 or MyD88 in Znrf1-/- mice attenuates these phenotypes, implying that ZNRF1 plays a potential role in preventing TLR7-dependent autoimmunity. Together, our data suggest that ZNRF1 negatively regulates TLR7/9 signaling, thereby decreasing the excessive production of type I IFNs and preventing the development of autoimmunity.