Genetic Analysis of Innate Immunity to Infection

巨噬细胞在许多病原体的早期免疫反应中是必不可少的。除了杀死病原体外，它们还可以通过将抗原呈递给T细胞来启动适应性免疫反应。干扰素-γ(IFNy)和肿瘤坏死因子(TNF)是成功清除多种感染性病原体所必需的细胞因子。巨噬细胞在这些细胞因子的协同作用下被激活，导致许多能杀死病原体或抑制其生长的效应分子上调。为了制定策略以提高对各种病原体的早期防御能力，了解个体对病原体的反应中的遗传变异是很重要的。我们的假设是，许多抗病基因的差异是由于巨噬细胞对病原体的反应不同和/或IFNy+肿瘤坏死因子的影响。因此，我们的目标是确定介导巨噬细胞对IFNy+肿瘤坏死因子和生物恐怖分子感染反应变化的基因。为了做到这一点，我们将测量重组近交系小鼠的IFNY+肿瘤坏死因子刺激或感染的巨噬细胞的转录组，这些重组近交系小鼠来自病原体敏感和抗性小鼠的杂交。然后，我们将使用数量性状基因座(QTL)分析来确定影响单个基因转录水平的小鼠基因组区域。幼稚或IFNY+肿瘤坏死因子刺激的巨噬细胞也将被各种病原体感染，并检测其抑制病原体生长的能力。影响巨噬细胞反应的基因组区域将与先前确定的影响抗病能力的基因组区域进行比较。确定表达QTL和疾病性状QTL的共同染色体位置将被用来提名疾病易感基因。这些将在体外杀伤试验中进行测试，方法是使用RNAi敲除基因，并测量对巨噬细胞杀死病原体能力的影响。我们将分别使用弓形虫和图拉氏弗朗西斯杆菌，NIAID B类和A类生物恐怖分子，它们在寄生虫性液泡和细胞质中复制。我们希望更深入地了解宿主对具有独特细胞内生活方式的病原体做出反应的遗传变异的分子基础。

Macrophages are essential in the early immune response against many pathogens. Besides killing pathogens they can also initiate the adaptive immune response by presenting antigens to T cells. Interferon gamma (IFNy) and tumor necrosis factor (TNF) are essential cytokines for successful clearance of many infectious agents. Activation of macrophages by synergistic effects of these cytokines leads to upregulation of many effector molecules that can kill pathogens or inhibit their growth. To devise strategies to improve the early defense against a variety of pathogens it is important to understand individual genetic variation in the response to pathogens. Our hypothesis is that many genetic differences in disease resistance are due to differences in the macrophage response to pathogens and/or to the effects of IFNy+TNF. Our goal is therefore to identify the genes mediating the variation in the macrophage response to IFNy+TNF and to infection with bioterrorism agents. To do this we will measure the transcriptome of IFNy+TNF-stimulated or infected macrophages from recombinant inbred mice derived from crosses between pathogen susceptible and resistant mice. We will then identify mouse genomic regions that affect transcription levels of individual genes using quantitative trait locus (QTL) analysis. Naive or IFNy+TNF-stimulated macrophages will also be infected with various pathogens and assayed for ability to inhibit the pathogen's growth. Genomic regions affecting the macrophage response will be compared to previously identified genomic regions affecting disease resistance. The identification of a common chromosomal location for both expression QTLs and disease trait QTLs will be used to nominate genes in the disease susceptibility locus. These will be tested in an in vitro killing assay by knocking down the gene using RNAi and measuring the effect on the macrophage's ability to kill the pathogen. We will use Toxoplasma gondii and Francisella tularensis, NIAID category B and A bioterrorism agents that replicate in a parasitophorous vacuole vs the cytoplasm, respectively. We expect to gain a deeper understanding of the molecular basis for genetic variation in the host response to pathogens with a distinct intracellular lifestyle.