Whole organism transcriptional profiling of innate immune response

描述(申请人提供)：脊椎动物的免疫反应由多个分子和细胞成分组成，这些成分必须相互作用，为宿主物种提供足够的防御病原体。虽然关于单个分子或细胞如何对感染做出反应的信息很多，但由于免疫系统的动态复杂性及其相互依赖的固有和适应功能，对整个有机体对病原体暴露的反应的完整了解仍然没有得到解决。斑马鱼幼体为克服这一障碍提供了一个独特的模型，因为幼体成功地防御病原体，同时在生命的头4-6周缺乏功能性适应性免疫系统，使得有可能在整个有机体的背景下专门检查先天免疫反应。在初步的转录图谱研究中发现，在斑马鱼幼体中对病原体相关分子模式做出反应的新基因，在成年斑马鱼和小鼠中也对感染刺激做出反应，这突显了这种新的先天免疫模型在基因发现方面的实用价值。据推测，斑马鱼幼体对感染刺激的转录反应将揭示介导哺乳动物先天免疫的新基因。为了验证这一假说，将达到两个目的，1)一种新的斑马鱼幼虫实验将被用来确定在没有适应性免疫的情况下整个生物体对病原体刺激的转录反应，以及2)功能未知的病原体反应基因在斑马鱼和哺乳动物的先天免疫反应中的作用。这项研究的长期目标是在整个有机体的背景下，在多个尺度上，从单个基因到转录组，建立一个综合的先天免疫反应模型。拟议实验的成功完成将促进未来的新研究，包括在体内显示透明斑马鱼幼体的感染反应，基于全生物体的化学遗传和突变筛选以确定免疫反应的药理和遗传修饰物，以及评估这些免疫反应基因在斑马鱼和哺乳动物的免疫防御和恢复中的作用的基因敲除/敲除研究。这些数据、试剂和工具将构成一个新的基于系统的、多生物体的范例，用于研究、评估和调节脊椎动物的先天免疫反应。 公共卫生相关性脊椎动物在机体水平上对感染作出反应的完整、动态的过程尚未被很好地理解。这项拟议的研究将利用一种新的策略来表征斑马鱼对免疫刺激的基因组反应，并利用这些信息来识别对病原体暴露做出反应的未表征的哺乳动物基因。这些方法产生的数据将为基于有机体的基因组感染反应提供前所未有的洞察力，促进调节免疫反应的新诊断、治疗和药物发现策略。

DESCRIPTION (provided by applicant): The vertebrate immune response is comprised of multiple molecular and cellular components that must interface to provide the host species with an adequate defense against pathogens. Although much information is available on how individual molecules or cells respond to infection, a complete understanding of the whole-organism response to pathogen exposure remains unresolved, due to the dynamic complexity of the immune system and its interdependent innate and adaptive functionality. The zebrafish larva provides a unique model for overcoming this obstacle as the larva successfully defends itself from pathogens while lacking a functional adaptive immune system for the first 4-6 weeks of life, making it possible to examine exclusively the innate immune response in a whole-organism context. In preliminary transcriptional profiling studies it was found that novel genes that respond to pathogen associated molecular patterns in the zebrafish larva, also respond to infection stimuli in adult zebrafish and mice, underscoring the utility of this novel innate immune model for gene discovery. It is hypothesized that the transcriptional response of zebrafish larvae to infection stimuli will reveal novel genes that mediate innate immunity in mammals. In order to test this hypothesis, two Aims will be achieved, 1) a novel zebrafish larvae assay will be employed to determine the whole-organism transcriptional response to pathogen stimuli in the absence of adaptive immunity, and 2) the role of functionally uncharacterized pathogen-responsive genes will be validated in zebrafish and mammalian innate immune response. The long range goal of the proposed research is to develop an integrative model of innate immune response in the context of the whole organism, on multiple scales, from the individual gene to the transcriptome. Successful completion of the proposed experiments will facilitate novel future investigations, including in vivo visualization of infection response in the transparent zebrafish larva, whole-organism based chemical genetic and mutagenesis screens to identify pharmacological and genetic modifiers of immune response, and gene knock-out/knock- down studies for assessing the role of these immune response genes in immune defense and recovery in both zebrafish and mammals. These data, reagents and tools will constitute a novel systems-based, multi- organism paradigm for investigating, evaluating and modulating the vertebrate innate immune response. PUBLIC HEALTH RELEVANCE The complete, dynamic process by which a vertebrate responds to infection on the organismal level is not well understood. The proposed research will utilize a novel strategy to characterize the genomic response of zebrafish to immune stimuli and use this information to identify uncharacterized mammalian genes that respond to pathogen exposure. The data generated by these methods will provide unprecedented insight into the organism based genomic response to infection, facilitating novel diagnosis, treatment and drug discovery strategies for modulating immune response.