Genetic Analysis of Innate Immunity to Infection

巨噬细胞在对抗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，它们还可以通过向T细胞呈递抗原来启动适应性免疫反应。干扰素γ (IFNy)和肿瘤坏死因子（TNF）是成功清除许多感染因子必不可少的细胞因子。通过这些细胞因子的协同作用激活巨噬细胞，导致许多效应分子的上调，这些效应分子可以杀死病原体或抑制其生长。为了设计策略来提高对各种病原体的早期防御，了解对病原体反应的个体遗传变异是很重要的。我们的假设是，疾病抵抗的许多遗传差异是由于巨噬细胞对病原体的反应和/或IFNy+TNF作用的差异。因此，我们的目标是确定介导巨噬细胞对IFNy+TNF和生物恐怖制剂感染反应变异的基因。为了做到这一点，我们将测量IFNy+ tnf刺激或感染的巨噬细胞的转录组，这些巨噬细胞来自病原体易感和抗性小鼠杂交的重组近交系小鼠。然后，我们将使用数量性状位点（QTL）分析确定影响单个基因转录水平的小鼠基因组区域。幼稚或IFNy+ tnf刺激的巨噬细胞也会被各种病原体感染，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疾病抗性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用于指定疾病易感位点上的基因。这些将在体外杀伤实验中进行测试，通过使用RNAi敲除基因并测量巨噬细胞杀死病原体的能力的影响。我们将使用刚地弓形虫和土拉弗朗西斯菌，NIAID B类和A类生物恐怖剂，分别在寄生液泡和细胞质中复制。我们期望对宿主对具有独特细胞内生活方式的病原体反应的遗传变异的分子基础有更深入的了解。

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.