Post-transcriptional regulation of the Dendritic Cell transcriptome

描述（由申请人提供）：适当控制免疫系统对人体健康至关重要。过敏的免疫系统会导致过敏和自身免疫性疾病，而免疫系统的损伤会使个体容易感染甚至癌症。树突状细胞（DC）在确保免疫系统的适当调节中起重要作用。针对病原体，DC迅速改变其表型以限制感染的传播，并启动适应性免疫应答。DC还通过维持自身耐受性参与预防自身免疫。现在理解的是，DC不是同质的细胞群体。它们由功能不同的亚群组成，具有不同的能力来处理抗原，对环境刺激做出反应，并参与不同的效应淋巴细胞。DC亚群包括专门将细胞相关抗原交叉呈递给CD8+ T细胞的CD8+ DC，以及浆细胞样DC（pDC），其有助于通过快速分泌大量干扰素α来建立抗病毒状态。 虽然我们现在认识到DC是一个异质性群体，但对驱动DC谱系定型和分化的机制以及控制功能特化的机制知之甚少。我们的初步分析数据和功能分析使我们假设特定的microRNA（miRNA）在调节DC身份中起关键作用。事实上，当我们特异性地敲除DC中的单个miRNA时，我们观察到小鼠中pDC的数量显著减少，但不是其他子集，并且pDC对TLR刺激的功能应答显著减少。在这里，我们提出了旨在更好地理解这种miRNA如何控制pDC发育和功能的研究，包括鉴定这种miRNA的相关靶点的研究（目的1）。我们还将研究我们通过分析研究鉴定的另外两种miRNA在DC亚群之间差异表达的作用（目的2）。为此，我们将使用我们产生的最先进的miRNA诱饵载体系统，该系统能够稳定抑制体内小鼠DC和培养的人DC中的miRNA。最后，我们将解决一个更广泛的问题，即如何使用我们开发的一种新的功能性检测方法在DC中调节miRNA本身，该方法允许高分辨率评估每个miRNA的细胞活性（目的3）。 我们已经集中精力在引起的作用，小的非编码RNA在DC中，因为这些小的非编码RNA是已知的，在控制细胞的身份发挥作用，因为相关的miRNA的发现可以用来揭示其他基因和途径，是重要的DC功能，通过识别的miRNA的调控目标。因此，我们的研究不仅将为特定miRNAs的作用提供新的见解，而且还将揭示参与DCs调控的其他基因。反过来，这将有助于找到免疫功能障碍的潜在新原因，并提供新的策略来增强或抑制免疫反应，以治疗疾病，并帮助疫苗开发。

DESCRIPTION (provided by applicant): Proper control of the immune system is critical to human health. A hypersensitive immune system can lead to allergies and autoimmune disease, whereas impairment of the immune system leaves an individual susceptible to infections and even cancers. Dendritic cells (DCs) play an important role in ensuring proper regulation of the immune system. In response to pathogens, DCs rapidly alter their phenotype to limit the spread of infection, and initiate an adaptive immune response. DCs are also involved in preventing autoimmunity by maintaining self tolerance. It is now understood that DCs are not a homogeneous population of cells. They are comprised of functionally distinct subsets with different abilities to process antigens, respond to environmental stimuli, and engage distinct effector lymphocytes. DC subsets include the CD8+ DCs that are specialized in the cross-presentation of cell associated antigens to CD8+ T cells, and plasmacytoid DC (pDC), which help to establish an antiviral state through the rapid secretion of high amounts of interferon alpha. While we now appreciate that DCs are a heterogeneous population, little is known about the mechanisms that drive DC lineage commitment and differentiation, as well as the mechanisms that control functional specialization. Our preliminary profiling data and functional analysis have led us to hypothesize that specific microRNA (miRNA) play a key role in regulating DC identity. Indeed, when we knocked out a single miRNA specifically in DCs we observed a significant reduction in the number of pDCs, but not other subsets in mice, and the pDCs had a major reduction in their functional response to TLR stimulation. Here, we propose studies aimed at better understanding how this miRNA controls pDC development and function, including studies to identify the relevant targets of this miRNA (Aim 1). We will also investigate the role of two other miRNA that we identified through profiling studies to be differentially expressed between DC subsets (Aim 2). To do this, we will use a state-of-the-art miRNA decoy vector system that we generated, which enables stable inhibition of a miRNA in mouse DCs in vivo and in human DCs in culture. Finally, we will address a broader question of how miRNAs themselves are regulated in DCs using a novel, functional assay that we developed, which permits high resolution assessment of each miRNA's cellular activity (Aim 3). We have focused our efforts on eliciting the role of miRNAs in DCs because these small non-coding RNAs are known to play a role in controlling cell identity, and because the discovery of relevant miRNAs can be used to uncover other genes and pathways that are important to DC function through the identification of the miRNA's regulatory targets. Thus, our studies will not only provide new insight into the role of specific miRNAs, but they will also reveal other genes involved in the regulation of DCs. This, in turn, will help to find potential new causes of immune dysfunction, and to supply new strategies to enhance or subdue immune responses for the treatment of diseases, and in the aid of vaccine development.