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。过度敏感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受到感染甚至癌症的影响。树突状细胞（dc）在确保免疫系统的适当调节中起着重要作用。在对病原体的反应中，dc迅速改变其表型以限制感染的传播，并启动适应性免疫反应。dc还通过维持自身耐受性参与预防自身免疫。现在了解到树突状细胞不是一个均匀的细胞群。它们由功能不同的亚群组成，具有不同的处理抗原、对环境刺激作出反应和参与不同效应淋巴细胞的能力。DC亚群包括CD8+ DC，它们专门向CD8+ T细胞交叉呈递细胞相关抗原，以及浆细胞样DC (pDC)，它们通过快速分泌大量α干扰素来帮助建立抗病毒状态。虽然我们现在认识到DC是一个异质种群，但我们对驱动DC谱系承诺和分化的机制以及控制功能专门化的机制知之甚少。我们的初步分析数据和功能分析使我们假设特异性microRNA （miRNA）在调节DC身份中起关键作用。事实上，当我们在dc中特异性敲除单个miRNA时，我们观察到pDCs的数量显著减少，但在小鼠中没有其他亚群，并且pDCs对TLR刺激的功能反应显著减少。在这里，我们提出了旨在更好地了解该miRNA如何控制pDC发育和功能的研究，包括确定该miRNA的相关靶点的研究（目的1）。我们还将研究另外两种miRNA的作用，我们通过分析研究确定了DC亚群之间的差异表达（目的2）。为了做到这一点，我们将使用我们开发的最先进的miRNA诱饵载体系统，该系统可以稳定地抑制小鼠dc和培养的人类dc中的miRNA。最后，我们将解决一个更广泛的问题，即miRNA本身是如何在DCs中被调节的，使用我们开发的一种新颖的功能分析方法，该方法允许对每种miRNA的细胞活性进行高分辨率评估（目的3）。我们一直致力于揭示miRNA在DC中的作用，因为已知这些小的非编码rna在控制细胞身份中起作用，并且因为相关miRNA的发现可用于通过鉴定miRNA的调控靶点来发现对DC功能重要的其他基因和途径。因此，我们的研究不仅将为特定mirna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，这将有助于发现免疫功能障碍的潜在新原因，并为疾病治疗提供增强或抑制免疫反应的新策略，并有助于疫苗开发。

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.