Innate Immune Activation in Malaria

描述（由申请人提供）：尽管新的有效的公共卫生战略已经明显降低了发病率和死亡率，但疟疾仍然是世界范围内一个严重的医学问题。这种疾病的消毒疫苗还没有到位，也许是因为我们对疟原虫免疫还有很多不了解。本申请是为了更新在疟疾先天免疫中的高产RO 1。我们的总体假设是，对疟疾的先天免疫反应是由两种高度协同的寄生产物驱动的：疟疾晶体疟原虫色素（Hz）和疟原虫DNA。我们的证据表明，DNA通过三种主要途径进入先天免疫细胞：在完整的寄生虫（在进入时可能是活的，也可能不是活的），在Hz的表面，或作为免疫复合物的一部分。总之，这些“病原体相关分子模式”（PAMPS）接合多种内溶酶体和胞质核苷酸受体，包括（但不限于）TLR 9。此外，疟原虫衍生的PAMP激活多种炎性体复合物，包括NLRP 3、NLRP 12和AIM 2。为了更好地描述对疟原虫感染的反应，我们提出了三个具体目标。 第一个目标是基于我们的假设，即胞质DNA受体在感染过程中被激活。这些受体出现，在一定程度上，通过一个独特的AT-丰富的基序，我们最近的特点是识别寄生虫DNA。我们建议使用多种方法的组合来探索这一假设，首先关注我们最近产生的四种基因敲除小鼠[CNBP，Mtr 4/Skiv 2L 2，IFI 16和环鸟苷一磷酸腺苷（cGAMP）合酶（cGAS）]。我们还制定了LSMS/MS方法，用于检测从疟疾患者血液中纯化的单核细胞产生cGAMP。发热患者中cGAMP的鉴定将是人类疾病中存在DNA驱动的先天免疫应答的有力证据。 目的2基于炎性小体驱动疟疾发病机制的假设。这些研究大多是在小鼠或小鼠来源的细胞中进行的，尽管我们在发热患者的细胞中也有良好的初步数据。我们首先建议在人类细胞和细胞系中使用功能丧失方法来扩展这些研究。更重要的是，我们将从患者的吞噬细胞中纯化炎性小体，并对这些复合物进行蛋白质组学分析，以公正的努力找到尚未意识到参与疾病的炎性小体。最后，我们建议量化表达组装炎性小体的细胞数量，并将这些发现与焦亡细胞死亡联系起来，因为我们的初步数据表明焦亡可能是广泛的（高达25%的循环单核细胞）。 在我们的最终目标中，我们将测试这样的假设，即疟疾中的炎症被调节到以前未被长非编码RNAS（lincRNA）识别的程度，类似于对LPS和其他TLR配体的反应。我们将开始关注lncRNA-Cox 2，它在小鼠中全面控制对TLR配体的反应。我们将进行RNA测序分析，以确定新的lincRNA，使用疟原虫PAMP体外刺激的细胞和从患者身上收集的细胞，以确定lincRNA实际上涉及疟疾。最后，我们将使用已建立的技术鉴定所鉴定的lncRNA的蛋白质结合伴侣和基因组靶标。我们相信，这项雄心勃勃的研究计划将大大增加我们对先天免疫的理解， 疟疾的研究，并有望产生新的方法和疫苗战略，可用于减少全球疾病负担。

DESCRIPTION (provided by applicant): Malaria continues to be a profound medical problem world wide, despite new and effective public health strategies that have clearly diminished morbidity and mortality. A sterilizing vaccine for the disease is still not within site, perhaps because there is still much that we do not know concerning plasmodial immunity. This application is to renew a highly productive RO1 on innate immunity in malaria. Our overall hypothesis is that the innate immune response to malaria is driven by two highly synergistic parasitic products: the malarial crystal hemozoin (Hz) and plasmodial DNA. Our evidence suggests that DNA gets into innate immune cells via three major routes: within the intact parasite (which may or may not be alive at the time of entry), on the surface of Hz, or as part of an immune complex. Together, these "Pathogen-associated molecular patterns" (PAMPS) engage a variety of endolysosomal and cytosolic nucleotide receptors, including (but not limited to) TLR9. In addition, plasmodial derived PAMPs activate multiple inflammasome complexes, including NLRP3, NLRP12 and AIM2. In order to better characterize the responses to plasmodial infections, we propose three specific aims. The first aim is based on our hypothesis that cytosolic DNA receptors are activated during infection. These receptors appear, in part, to recognize parasite DNA via a unique AT-rich motif that we have recently characterized. We propose to explore this hypothesis using a combination of approaches, focusing first on four knockout mice that we have recently generated [CNBP, Mtr4/Skiv2L2, IFI16 and the enzyme cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS)]. We have also worked out LSMS/MS methodologies for examining the production of cGAMP from monocytes purified from the blood of malaria patients. The identification of cGAMP in febrile patients would be strong evidence that there is a DNA driven innate immune response in human disease. Aim 2 is based on the hypothesis that inflammasomes drive the pathogenesis of malaria. Most of these studies have been performed in mice, or mouse-derived cells, although we have good preliminary data in cells from febrile patients as well. We first propose to extend these studies using loss of function approaches in human cells and cell lines. More importantly, we will purify inflammasomes from the phagocytic cells of patients and subject these complexes to proteomic analysis, in an unbiased effort to find identify inflammasomes not yet realized to be in involved in disease. Finally, we propose to quantify the number of cells expressing assembled inflammasomes and relate these findings to pyroptotic cell death, as our preliminary data suggest that pyroptosis may be extensive (up to 25% of circulating monocytes). In our final Aim, we will test the hypothesis that inflammation in malaria is regulated to a degree not previously recognized by long non-coding RNAS (lincRNAs), similar to the response to LPS and other TLR ligands. We will begin by focusing on lncRNA-Cox2, which globally controls responses to TLR ligands in mice. We will perform RNA-sequencing analysis to identify novel lincRNAs using cells stimulated in vitro with plasmodial PAMPs and cells harvested from patients, to identify lincRNAs actually involved in malaria. Finally, we will identify protein bindng partners and genomic targets of identified lncRNAs using established techniques. We believe that this ambitious research plan will add significantly to our understanding of innate immunity in malaria and in hopefully give rise to novel approaches and vaccine strategies that can be used to reduce the global burden of disease.