ZCCHC6在調節TLR4引發Interleukin-10生成的作用機制

Abstract

先天性免疫系統是生物體防禦病原體入侵的第一道防線。先天免疫反應失調與許多病原體感染和發炎的疾病息息相關。因此，先天免疫反應必須受到嚴謹的調控。Toll like receptors (TLRs)是先天免疫系統中的其中一個重要的成員。一些由TLRs所介導的免疫反應的修飾作用已被報導過，除了已經被廣泛研究其參與在免疫訊息傳遞的磷酸化作用及泛素化作用之外，後轉錄調控是一種能快速且有彈性地調節免疫和發炎反應的方式。但是在TLR所誘導的免疫反應中的後轉錄調控，其參與的機制仍需要進一步被探討。我們先前發現一個terminal uridyltransferase (TUTase)的成員，ZCCHC6，其表現量在TLR配體刺激下有上升的趨勢。ZCCHC6已知具有在miRNA或是mRNA末端接上尿苷酸的功能，進而促使RNA的穩定或是降解。在Zcchc6缺失的巨噬細胞中，抗發炎細胞激素Il10的mRNA表現量於一個TLR4配體，LPS，的誘導下顯著地提升。在我們的研究中，我們欲闡明ZCCHC6調控IL-10表現量的詳細機制。我們首先證實在LPS的刺激下，Il10 mRNA及蛋白質表現量在Zcchc6缺失的RAW 264.7巨噬細胞及小鼠骨髓分化的巨噬細胞(BMDMs)中皆被誘升。此外，在LPS刺激下，一個在Il10啟動子裡的假定的STAT結合基序(putative STAT binding motif)會藉由被STAT1結合而參與在ZCCHC6所調控的Il10轉錄作用中。同時，我們也發現在Zcchc6缺失的巨噬細胞中，LPS增強了STAT1的表現量。ZCCHC6可能藉由辨識在Stat1 3’端非轉譯區上的莖環結構來調節Stat1 mRNA的穩定性。另一方面，我們的研究顯示，STAT2的表現量在Zcchc6缺失及LPS活化下有增加的現象。在LPS誘導下，STAT1和STAT2可能形成二聚體，並結合至在Il10啟動子裡的假定的STAT結合基序，但是其中ZCCHC6是如何調控STAT2的機制仍需再被更深入地研究。综上所述，我們的研究證實ZCCHC6會藉由調節Stat1 mRNA的穩定性，而使得STAT1結合到Il10啟動子上的假定的STAT結合基序減少，來達到調控LPS所誘導的IL-10的表現量。作為一個抗發炎反應的細胞激素，IL-10在為了因應病原體的侵襲而平衡發炎反應上扮演很重要的角色。因此，了解ZCCHC6如何調節IL-10的表現量能進一步幫助我們擬定對應發炎疾病的治療策略。

The innate immune response is the first line of defense against pathogen infection. Dysregulation of the innate immune response has been closely associated with the pathogenesis of numerous infectious and inflammatory disorders. Therefore, the innate immune response must be tightly controlled. Toll like receptors (TLRs) are one of the key players in the innate immune system. Several modifications in TLR-mediated immune responses have been reported. In addition to phosphorylation and ubiquitination, which have been widely investigated for their roles in immunological signaling, post-transcriptional regulation is a quick and flexibly way to modulate the immune and inflammatory responses. However, the underlying mechanisms of the post-transcriptional modifications involved in TLR-triggered immune responses remain to be fully elucidated. We previously identified a terminal uridyltransferase (TUTase), ZCCHC6, which has been shown to uridylate miRNAs and mRNAs and further promote the stabilization or degradation of RNAs, was upregulated upon TLR ligands treatment. Anti-inflammatory cytokine, IL-10, mRNA expression was significantly increased in response to LPS, a TLR4 ligand, in Zcchc6-depeleted macrophages. In this study, we aimed to elucidate the detailed mechanism how ZCCHC6 regulates IL-10 expression. We first confirmed that the levels of Il10 mRNA and protein were induced in both Zcchc6-depleted RAW 264.7 macrophages and bone marrow-derived macrophages (BMDMs) after LPS challenge. In addition, we identified a novel putative STAT binding motif in the Il10 promoter, which was responsible for ZCCHC6-mediated Il10 transcription after LPS stimulation via binding by STAT1. Furthermore, STAT1 expression was increased in Zcchc6-depleted macrophages, and its mRNA stability was regulated by ZCCHC6 possibly through recognizing the stem-loop structures in Stat1 3’UTR. We also revealed that STAT2 expression was increased in Zcchc6-deficient macrophages. STAT1 and STAT2 possibly formed a dimer and bound to the putative STAT binding motif in the Il10 promoter after LPS treatment, but, the underlying mechanism by which ZCCHC6 regulates STAT2 expression remains to be further elucidated. Taken together, our findings suggest that ZCCHC6 regulates TLR4-activated Il10 transcription through modulating the mRNA stability of the transcription factor Stat1, leading to the reduction of the binding of STAT1 to the putative STAT binding element in the Il10 promoter. Given the importance of IL-10 in counteracting inflammatory responses, understanding how ZCCHC6 modulates IL-10 expression may pave a way to provide a therapeutic strategy for many inflammatory diseases.