E3泛素連接酶ZNRF1調控TLR7及TLR9訊息傳遞之研究

Abstract

漿狀樹突細胞 (plasmacytoid dendritic cells)是哺乳類先天免疫系統中一群扮演哨兵角色的細胞，它們高度表現第七型及第九型類鐸受體 (Toll-like receptor 7 and 9)，此二類鐸受體能分別偵測單股核糖核酸 (single-strand RNA)及未甲基化的CpG位點去氧核糖核酸 (CpG DNA)。當第七型及第九型類鐸受體偵測到配體後，漿狀樹突細胞會立即產生大量的第一型干擾素 (type Ι interferons)來抑制病毒的複製並且促進周圍細胞的抗病毒功能。ZNRF1 (Zinc and ring finger 1)是一種E3泛素連接酶，過去被報導在瓦氏退化現象 (Wallerian degeneration)中扮演重要角色。我們實驗室先前的研究發現ZNRF1在第四型類鐸受體 (Toll-like receptor 4)的活化下會增加促發炎細胞激素 (pro-inflammatory cytokine)並減少抗發炎細胞激素中的白血球介白素-10 (interlukin 10 )，顯示ZNRF1在第四型類鐸受體所誘導的發炎反應中扮演正向調節的角色。我們也進一步發現，當類FMS的酪氨酸激酶-3受體所分化的漿狀樹突細胞(Flt3L-driven pDCs)及人類的漿狀樹突細胞株 (CAL-1)中的第七型及第九型類鐸受體活化後，ZNRF1缺失會增強第一型干擾素產生。在這篇研究中，我們試圖去探討在第七型及第九型類鐸受體所誘導的免疫反應中，ZNRF1的詳細調控機制以及在此過程中ZNRF1是如何被活化。我們首先確認用第七型及第九型類鐸配體刺激人類的漿狀樹突細胞株 (CAL-1)後，ZNRF1缺失會增強第一型干擾素產生。在ZNRF1缺失的人類漿狀樹突細胞株 (CAL-1)中，第七型類鐸受體的活化會增加IKKα、MAPK及IKKβ/NF-κB的活化，而第九型類鐸受體的活化僅會增加的活化。此外，在ZNRF1 103號的酪氨酸對於第七型及第九型類鐸受體所誘導的免疫反應是重要的。我們的動物實驗結果顯示在流感病毒H5N1的感染下IKKα，ZNRF1缺失的小鼠相較於野生型小鼠表現更高的存活率。在流感病毒H5N1感染後第六天，ZNRF1缺失小鼠肺臟中的促發炎細胞激素、第一型干擾素、趨化介素及病毒的mRNA都有較低的情形。然而，究竟ZNRF1缺失在小鼠身上會影響病毒的清除還是細胞激素的產生仍有待之後的實驗釐清。總結以上，我們的結果顯示在漿狀樹突細胞中，ZNRF1可能會透過控制類鐸受體的運輸而負向調節第七型及第九型類鐸受體所驅動的免疫反應。

Plasmacytoid dendritic cells (pDCs), a unique sentinel cell population in the mammalian innate immunity system, express high levels of endosomal Toll-like receptors (TLRs) TLR7 and TLR9, which sense single-strand RNA (ssRNA) and unmethylated CpG DNA, respectively. Upon engagement of TLR7 and TLR9, pDCs produce massive quantities of type Ι interferons (IFNs) rapidly to inhibit viral replication and promote the antiviral functions in surrounding cells. Zinc and ring finger 1 (ZNRF1) is an E3 ubiquitin ligase, which plays a crucial role in Wallerian degeneration. Previous study in our lab demonstrated that ZNRF1 enhanced the production of pro-inflammatory cytokines and reduced anti-inflammatory cytokine IL-10 upon TLR4 activation, suggesting its positive regulatory role in TLR4-triggered inflammatory response. We further demonstrated that ZNRF1 depletion enhanced type  interferons in both FMS-like tyrosine kinase 3 ligand (Flt3L)-driven pDCs and CAL-1 cells (human pDCs) upon TLR7 and TLR9 activation. In this study, we aim to reveal the detailed mechanism by which ZNRF1 regulates TLR7- and TLR9-mediated immune responses and how ZNRF1 is activated upon engagement of TLR7 and TLR9. We confirmed that ZNRF1 depletion enhanced type Ι interferon production in CAL-1 cells after stimulation of TLR7 and TLR9 ligands. Activation of IKKα, MAPKs and IKKβ/NF-κB was increased in ZNRF1-depeleted CAL-1 cells after TLR7 engagement. However, only increased IKKα activation was observed in ZNRF1 depletion cells after TLR9 activation. In addition, tyrosine phosphorylation site ZNRF1 on residue 103 is required for TLR7- and TLR9-mediated immune responses. Furthermore, our results showed that ZNRF1 knockout mice displayed better survival compared with wildtype mice after challenged with H5N1 influenza virus (IAV). Lower levels of pro-inflammatory cytokine, type Ι interferons, chemokines and viral mRNA were decreased in Znrf1-/- lungs at day 6 after intratrachea infection of H5N1 IAV. Nevertheless, it remains to be further investigated whether depletion of ZNRF1 in mice affects viral clearance or cytokine production. Taken together, our results suggest that ZNRF1 is a negative regulator of TLR7- and TLR9-driven immune response in pDCs possible through controlling receptor trafficking.