Innate Immune Activation in Malaria

描述（由申请人提供）：尽管新的和有效的公共卫生战略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疗问题。目前还没有针对这种疾病的消毒疫苗，也许是因为我们对疟原虫免疫还有很多不了解的地方。这一应用是为了更新疟疾先天免疫的高产RO1。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：疟疾晶体血色素（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：在完整的寄生虫体内（在进入时可能是活的，也可能不是活的），在Hz表面，或者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）与多种内溶酶体和胞质核苷酸受体结合，包括（但不限于）TLR9。此外，疟原虫衍生的PAMPs可激活多种炎性小体复合物，包括NLRP3、NLRP12和AIM2。为了更好地表征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受体在感染期间被激活。这些受体似乎在一定程度上通过我们最近描述的独特的富含at的基序来识别寄生虫的DNA。我们建议使用多种方法来探索这一假设，首先关注我们最近产生的四个基因敲除小鼠[CNBP， Mtr4/Skiv2L2， IFI16和环鸟苷单磷酸腺苷（cGAMP）合成酶（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。在发热患者中发现cGAMP将有力地证明在人类疾病中存在DNA驱动的先天免疫反应。目的2是基于炎症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很好的初步数据。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，以不偏不倚地寻找尚未意识到与疾病有关的炎性小体。最后，我们建议量化表达组装炎性小体的细胞数量，并将这些发现与热亡细胞死亡联系起来，因为我们的初步数据表明，热亡可能是广泛的（高达25%的循环单核细胞）。在我们的最终目标中，我们将验证疟疾炎症在一定程度上受到长链非编码rna （lincRNAs）的调节，类似于对LPS和其他TLR配体的反应。我们将首先关注lncRNA-Cox2，它全局控制小鼠对TLR配体的反应。我们将进行rna测序分析，利用体外受疟原虫PAMPs刺激的细胞和从患者身上采集的细胞来鉴定新的lincRNAs，以鉴定实际参与疟疾的lincRNAs。最后，我们将使用已建立的技术鉴定鉴定的lncrna的蛋白质结合伙伴和基因组靶点。我们相信这个雄心勃勃的研究计划将大大增加我们对先天性免疫的理解

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.