Macrophage-Activating MicroRNAs In the Pathogenesis of Leishmaniasis

描述（由申请人提供）： 利什曼原虫属原生动物对它们入侵的宿主细胞有着深远的影响。这些寄生虫通常存在于细胞内的巨噬细胞中，巨噬细胞是一种具有杀死细胞内微生物能力的细胞类型。利什曼原虫不是屈服，而是麻痹宿主细胞的杀微生物途径，将巨噬细胞从致死细胞变成细胞内的避风港，使寄生虫存活，复制并最终传播到邻近细胞。寄生虫“麻痹”宿主杀微生物功能的机制尚未完全了解。 最近发现，大多数真核生物利用短的非编码RNA序列来全局调节多种基因的表达。事实上，长度为18-30 bp的短RNA序列（称为microRNA）对于调节估计30%的人类基因组的表达至关重要。该提议的假设是microRNA是决定利什曼原虫感染后巨噬细胞活化模式的上游触发因子。我们将讨论以下问题。(1)在感染巨噬细胞的过程中，寄生虫通常会诱导或抑制哪些microRNA？(2)哪些microRNA被诱导以响应激活巨噬细胞朝向不同极性表型的刺激？(3)在目标1和2中发现的microRNA之间是否存在重叠，实验操作这些microRNA对利什曼病感染的巨噬细胞有何影响？该提案的具体目标是：1。为了对L. chagasi由人类巨噬细胞。2.使用类似的分析方法来确定哪些microRNA响应于巨噬细胞活化而被诱导向不同的极化表型。主要感兴趣的是在M1（经典）活化期间发生的microRNA变化，以及其他形式的巨噬细胞活化的相互变化，因为经典活化的巨噬细胞可以杀死细胞内的利什曼原虫。3.将目的1和2的结果相关联，并选择性地过表达或抑制巨噬细胞表达选定的microRNA，这些microRNA可能改变巨噬细胞活化或细胞内寄生虫生长的模式。 公共卫生相关性： 利什曼病在持久自由行动（OEF）或伊拉克自由行动（OIF）期间达到了流行病的程度，达到了二战以来美国军队从未经历过的水平。在2002-03年期间，在部署在伊拉克、科威特和阿富汗的美国士兵中诊断出600多例皮肤利什曼病和4例内脏利什曼病。尽管受到国际机构的关注，利什曼病仍然是一种难以预防和治疗的疾病。随着越来越多的部队从中东的驻地返回，利什曼病问题在退伍军人管理局系统中变得越来越普遍。该提案的目的是探索microRNA在利什曼病发病机制中的作用，认为操纵小的调节RNA代表了治疗这种和其他细胞内感染性疾病的创新未来途径。

DESCRIPTION (provided by applicant): The Leishmania spp. protozoa have a profound effect on the host cell that they invade. These parasites reside intracellularly usually in macrophages, a cell type with the capacity to kill intracellular microbes. Rather than succumb, Leishmania paralyze the microbicidal pathways of the host cell, changing the macrophage from a lethal cell to an intracellular safe haven which allows the parasite to survive, replicate and ultimately spread to neighboring cells. The mechanism(s) through which the parasite "paralyzes" the host microbicidal function is incompletely understood. It has recently come to light that most eukaryotic organisms utilize short noncoding RNA sequences to globally regulate expression of a wide variety of genes. Indeed short RNA sequences of 18-30 bp in length, called microRNAs, are critical for regulating expression of an estimated 30% of the human genome. The hypothesis underlying this proposal is that microRNAs are the upstream trigger(s) determining which pattern of macrophage activation will occur after Leishmania infection. We will address the following questions. (1) What microRNAs does the parasite usually induce or suppress during infection of macrophages? (2) Which microRNAs are induced in response to stimuli that activate macrophage toward different polar phenotypes? (3) Is there overlap between the microRNAs discovered in Aims 1 and 2, and what is the effect of experimental manipulation of these microRNAs on Leishmania-infected macrophages? Specific aims of the proposal are: 1. To perform a global profiling of changes in microRNA expression induced in response to phagocytosis of L. chagasi by human macrophages. 2. To use a similar profiling approach to determine which microRNAs are induced in response to macrophage activation toward different polarized phenotypes. Of primary interest will be the microRNA changes that occur during M1 (classical) activation with reciprocal changes in other forms of macrophage activation, since classically activated macrophages can kill intracellular Leishmania. 3. To correlate the results of Aims 1 and 2, and to selectively either overexpress or suppress macrophage expression of selected microRNAs that may change in the pattern of macrophage activation or intracellular parasite growth. PUBLIC HEALTH RELEVANCE: Leishmaniasis reached epidemic proportions during Operation Enduring Freedom (OEF) or Operation Iraqi Freedom (OIF), to a level not experienced by the US military since World War II. During 2002-03 there were more than 600 cases of cutaneous leishmaniasis and 4 cases of visceral leishmaniasis diagnosed in American soldiers deployed in Iraq, Kuwait and Afghanistan. Despite attention from international agencies, leishmaniasis remains a difficult disease to prevent and treat. The problem of leishmaniasis is becoming more prevalent in the VA system, as more troops return from stations in the Middle East. The purpose of this proposal is to explore the role of microRNAs in the pathogenesis of leishmaniasis, with the view that manipulation of small regulatory RNAs represents an innovative future avenue for therapy of this and other intracellular infectious diseases.