Innate Immune Activation in Malaria

描述（由申请人提供）：疟疾仍然是全世界疾病和死亡的主要原因。关于疟疾发病机制的两个主要未解决的问题是：1)寄生虫中的哪些分子激活先天免疫系统并引起炎症；2)由这些微生物产物连接的受体是什么。该项目的总体目标是确定恶性疟原虫感染个体全身炎症和发热的分子机制。我们的初步数据表明，疟疾DNA，特别是当与疟疾色素血色素结合时，会激活炎症。这一提议的基本假设是，急性疟疾的发烧和炎症是由疟疾DNA与吞噬细胞表达的DNA受体相互作用引起的。我们进一步假设，对疟疾DNA的先天免疫反应是由两个受体介导的：TLR9和一个尚未确定的富含at的DNA基序受体。这一假设挑战了疟疾糖基磷脂酰肌醇(GPI)-脂质锚定物（主要是TLR2/CD14激动剂）是疟疾全身性炎症的主要原因这一概念。为了验证这一假设，我们建议将对急性发热性疟疾患者细胞免疫激活的检查与体外工作结合起来。我们提出三个目标。第一个目标是确定疟疾诱导的先天免疫激活是否主要发生在TLR9+细胞中，还是主要发生在表达TLR2和/或CD14的细胞中。我们将在纯化细胞中分析基因诱导，并在培养和患者中定义TLR表达，以确定使用哪些受体和信号通路。从疟疾基因组中提取的DNA和寡核苷酸将与纯化的疟疾GPI锚点进行比较。在我们的第二个目标中，我们将评估血色素蛋白在炎症中的重要性。将收集疟疾患者的PBMC，将其分为血色素蛋白阳性和阴性的单核细胞和dc，并评估免疫激活的标志物。我们将对免疫细胞中的疟原虫色素和疟疾DNA贩运进行共聚焦成像研究。这些研究将确定贩运和先天免疫反应之间的时空关系，包括特定DNA受体的参与和炎性体的激活。在最后的Aim中，我们将重点关注从疟疾基因组中提取的富含at的寡核苷酸的免疫活性。我们将重点关注；1)那些导致I型干扰素产生的信号通路，我们发现这是小鼠脑型疟疾结果的关键决定因素；2)炎性小体的激活，主要通过NRLP3和AIM2，这是我们最近发现的炎性小体成分。信号通路将同时使用分子遗传学方法和蛋白质组学来建立。我们将在小鼠脑疟疾中测试炎性小体敲除小鼠，包括一种新的AIM2 KO，从而将我们的体外研究结果与体内疾病联系起来。我们的最终目标是确定疟疾DNA在疟疾感染期间介导先天免疫反应中的相对作用，并最终确定相关的DNA受体。

DESCRIPTION (provided by applicant): Malaria remains a major cause of illness and death worldwide. Two major unresolved issues concerning the pathogenesis of malaria are: 1) what molecules from the parasite activate the innate immune system and cause inflammation and, 2) what are the receptors that are ligated by these microbial products. The overall goal of this project is to identify the molecular mechanisms of systemic inflammation and fever in individuals infected with Plasmodium falciparum. Our preliminary data demonstrate that malaria DNA, especially when complexed to the malarial pigment hemozoin, activates inflammation. The underlying HYPOTHESIS of this proposal is that fever and inflammation in acute malaria are initiated by the interaction of malarial DNA with DNA receptors expressed by phagocytes. We further hypothesize that the innate immune response to malarial DNA is mediated by two receptors: TLR9 and an as yet unidentified receptor for AT-rich DNA motifs. This hypothesis challenges the concept that malarial glycosylphosphatidylinositol (GPI)-lipid anchors, which are primarily TLR2/CD14 agonists, are the primary cause of systemic inflammation in malaria. In order to test this hypothesis, we propose to combine an examination of immune activation in cells from patients with acute febrile malaria with in vitro work. We propose three Aims. The first Aim is to determine if innate immune activation induced by malaria, occurs primarily in TLR9+ cells, or in cells that primarily express TLR2 and/or CD14. We will profile gene induction in purified cells with defined TLR expression in culture and from patients in order to determine which receptors and signaling pathways are employed. DNA, and oligonucleotides derived from the malarial genome, will be compared to purified malarial GPI anchors. In our second Aim, we will assess the importance of hemozoin in inflammation. PBMC will be collected from malaria patients, sorted into hemozoin positive and negative monocytes and DCs, and assessed for markers of immune activation. We will perform confocal imaging studies of hemozoin and malarial DNA trafficking in immune cells. These studies will determine the temperospatial relationships between trafficking and innate immune responses, including the engagement of specific DNA receptors and the activation of the inflammasome. In the final Aim, we will focus on the immune activity of AT-rich oligonucleotides whose sequences are derived from the malarial genome. We will focus on; 1) those signaling pathways that result in the production of Type I interferons, which we have found to be a critical determinant of outcome in mouse cerebral malaria; and 2) activation of the inflammasome, primarily through NRLP3 and AIM2, an inflammasome component that we recently discovered. Signaling pathways will be established using both a molecular genetics approach and the use of proteomics. We will test inflammasome knockout mice, including a new AIM2 KO, in mouse cerebral malaria, thus relating our in vitro findings with in vivo disease. Our ultimate goal is to define the relative role of malarial DNA in mediating innate immune responses during malarial infection and to definitively identify related DNA receptors. PUBLIC HEALTH RELEVANCE: Malaria is the world's most common infectious disease, and kills millions of individuals annually. US citizens risk obtaining malaria when they travel or are engaged in military operations in tropical areas. The purpose of this grant is to gain a better understanding of why malaria causes disease in the hopes that better therapies can be devised, including an effective vaccine that might prevent malaria.