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，在许多国家，包括大多数中东国家高度流行。军事人员有感染这种病媒传播疾病的危险。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，利什曼病在美国军队中达到了流行病的程度。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更多未被识别的感染，而不是报告的约600人。 利什曼原虫即使在临床“治愈”后也有能力在宿主中持续存在。事实上，受感染的人类和其他宿主可能永远不会清除感染，即使在疾病症状消失后。已知利什曼原虫在感染期间对宿主的先天性和适应性免疫应答产生深远影响。最近的证据表明，即使在疾病“治愈”后，免疫效应仍然存在。因此，很可能利什曼病的免疫影响超出了可测量的症状性疾病。 外泌体是从大多数活细胞释放的小囊泡，其含有来自细胞来源的蛋白质、RNA和脂质。外泌体现在被认为是宿主细胞之间运输免疫和其他反应信号的主要血管。最近认识到外泌体是从利什曼原虫释放的。这些微小的信息包可能提供线索，解释利什曼原虫感染的深刻的系统性影响。 我们已经表征了来自婴儿利什曼原虫不同生命阶段的外泌体的蛋白质含量，婴儿利什曼原虫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，即来自利什曼原虫或利什曼原虫感染细胞的外泌体中释放的蛋白质和小RNA是利什曼原虫病对免疫细胞反应的戏剧性和持久性局部和全身作用的原因。这些效应可以在活动性或无症状感染期间引起。我们的推论假定，利什曼原虫自身释放外泌体，其内容物影响细胞内环境，并将自身并入从宿主细胞释放的外泌体中。因此，了解释放的外泌体的宿主和寄生虫来源的内容，将为诊断和治疗利什曼病的新方法提供基础。该项目的具体目标是：目标1。鉴定外泌体的蛋白质和小RNA含量，以及外泌体从感染L. major或L.婴儿假设：来自利什曼原虫感染的巨噬细胞的外来体含有促进感染的巨噬细胞的非经典活化的蛋白质和微RNA。目标2。鉴定来自在伊拉克获得皮肤利什曼病的美国军人的血清和尿液外泌体中的蛋白质和小RNA，并确定外泌体蛋白质是否在感染利什曼病的人类或小鼠中引起免疫应答。major或L.婴儿假设：从利什曼原虫感染的哺乳动物细胞释放的外泌体含有在感染个体中产生免疫应答的优势寄生虫抗原。目标3。记录来自寄生虫、感染的巨噬细胞或在利什曼病患者血清中循环的外泌体的免疫调节特性。将在人巨噬细胞和L. major和L.婴儿感染假设：外泌体表现出影响感染和旁观者宿主免疫细胞的活化状态和杀微生物反应的功能性酶活性。

 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.