The Role of Exosomes in the Pathogenesis of Leishmaniasis

 描述(由申请人提供)： 利什曼病是指一组常见的寄生虫病，在包括大多数中东国家在内的许多国家高度流行。军事人员面临着这种病媒传播疾病的风险。事实上，在持久自由行动(OEF)、伊拉克自由行动(OIF)和新黎明行动(OND)期间，利什曼病在美军中达到了流行的程度。在不同的研究中，无症状利什曼病与有症状利什曼病的比例从6：1到98：1不等。因此，我们的军事人员中很可能有比报告的~600人更多的未被识别的感染。即使在临床“治愈”之后，利什曼原虫也有能力在宿主体内坚持下去。事实上，被感染的人和其他宿主可能永远不会清除感染，即使在疾病症状消失之后。众所周知，利什曼原虫在感染期间对宿主的先天和获得性免疫反应产生深远影响。最近的证据表明，即使在疾病被“治愈”之后，免疫效应仍然存在。因此，利什曼病的免疫影响很可能超出了可测量的症状性疾病。外体是大多数活细胞释放的小囊泡，含有起源细胞的蛋白质、RNA和脂类。Exosome现在被认为是在宿主细胞之间传输免疫和其他反应信号的主要血管。最近，人们认识到外体是从利什曼原虫中释放出来的。这些微小的信息包可能提供解释利什曼原虫感染的深刻系统性影响的线索。我们已经确定了不同生活阶段婴儿利什曼原虫外体的蛋白质含量，这是中东利什曼病的病原体之一。目前的建议是基于这样的假设，即利什曼原虫的外体或从利什曼原虫感染的细胞中释放的蛋白质和小RNA是利什曼原虫对免疫细胞反应产生戏剧性和持久性的局部和全身影响的原因。这些效应可以在活动感染期间或在无症状感染期间调用。我们的推论是利什曼原虫自己释放外体，这些外体的内容影响细胞内环境，并被合并到从宿主细胞释放的外体中。因此，了解释放的外切体中宿主和寄生虫衍生的内容将为利什曼病的诊断和治疗提供新的方法基础。本项目的具体目标是：目的1.鉴定巨噬细胞感染巨噬细胞后外切体的蛋白质和小RNA含量，以及巨噬细胞释放外切体的途径。假设：利什曼病感染的巨噬细胞的外切体含有促进感染巨噬细胞非经典激活的蛋白质和microRNAs。目的#2.从在伊拉克感染皮肤利什曼病的美军成员中鉴定血清和尿液外切体中的蛋白质和小RNA，并确定外切体蛋白是否在人类或感染主要利什曼原虫或婴儿利什曼原虫的小鼠中引发免疫反应。假设：从感染利什曼原虫的哺乳动物细胞释放的外体含有主要的寄生虫抗原，这些抗原能在感染者身上产生免疫反应。目的#3.记录来自寄生虫、感染的巨噬细胞或人类利什曼病患者血清中循环的外切体的免疫调节特性。免疫反应将在人类巨噬细胞以及大乳杆菌和婴儿乳杆菌感染的小鼠模型中进行测试。假设：外切体表现出功能性的酶活性，影响感染和旁观者宿主免疫细胞的激活状态和杀菌反应。

 DESCRIPTION (provided by applicant): Leishmaniasis refers to a group of common parasitic diseases that are highly endemic in many nations including most Middle Eastern countries. Military personnel are at risk for this vector-borne disease. Indeed, leishmaniasis reached epidemic proportions in the US military during the Operations Enduring Freedom (OEF), Iraqi Freedom (OIF) and New Dawn (OND). The ratio of asymptomatic to symptomatic leishmaniasis varies from 6:1 to 98:1 in different studies. It is therefore highly likely that there are many more unrecognized infections in our military personnel than the ~600 reported. Leishmania have the ability to persist in a host even after clinical "cure". Indeed, infected humans and other hosts may never clear the infection, even after disease symptoms resolve. Leishmania are known to exert a profound effect on innate and adaptive immune responses of the host during infection. Recent evidence documents that immune effects persist even after "cure" of disease. Thus, it is highly likely that the immune effects of leishmaniasis extend beyond measurable symptomatic disease. Exosomes are small vesicles released from most living cells that contain protein, RNA and lipids from the cell of origin. Exosomes are now recognized as major vessels transporting immune and other response signals between host cells. Recently it was recognized that exosomes are released from Leishmania parasites. These tiny packages of information may provide clues that explain the profound systemic effects of Leishmania infection. We have already characterized the protein content of exosomes from different life stages of Leishmania infantum, one of the causative agents of leishmaniasis in the Middle East. The current proposal is based on the hypothesis that proteins and small RNAs released in exosomes from Leishmania or from Leishmania-infected cells are responsible for the dramatic and persistent local and systemic effects of leishmaniasis on immune cell responses. These effects could be invoked during active or during asymptomatic infection. Our corollary posits that Leishmania parasites themselves release exosomes whose contents affect the intracellular environment, and which become incorporated themselves into exosomes released from host cells. An understanding of the host- and parasite-derived content of released exosomes, therefore, would provide the basis for novel approaches to diagnosis and therapy of leishmaniasis. Specific aims of this project are: Aim #1. Identify the protein and small RNA content of exosomes, and the pathway of exosome release from macrophages infected with L. major or L. infantum. Hypothesis: Exosomes from leishmania- infected macrophages contain protein and microRNAs that promote non-classical activation of infected macrophages. Aim #2. Identify proteins and small RNAs in the serum and urine exosomes from members of the US military who acquired cutaneous leishmaniasis in Iraq, and determine whether exosome proteins elicit an immune response in humans or in mice infected with L. major or L. infantum. Hypothesis: Exosomes released from Leishmania-infected mammalian cells contain dominant parasite antigens that generate an immune response in the infected individual. Aim #3. Document the immunomodulatory properties of exosomes from parasites, from infected macrophages, or circulating in serum of humans with leishmaniasis. Immune responses will be tested in human macrophages and in mouse models of L. major and L. infantum infection. Hypothesis: Exosomes exhibit functional enzymatic activities that affect the activation state and the microbicidal response of both the infected and bystander host immune cells.