Whole organism transcriptional profiling of innate immune response

描述（由申请方提供）：脊椎动物免疫应答由多种分子和细胞组分组成，这些组分必须相互作用，为宿主物种提供足够的病原体防御。虽然有很多关于单个分子或细胞如何对感染作出反应的信息，但由于免疫系统的动态复杂性及其相互依赖的先天和适应性功能，对整个生物体对病原体暴露的反应的完整理解仍然没有得到解决。斑马鱼幼虫为克服这一障碍提供了一个独特的模型，因为幼虫在生命的前4-6周缺乏功能性适应性免疫系统的同时成功地保护自己免受病原体的侵害，这使得在整个生物体背景下专门检查先天免疫反应成为可能。在初步的转录谱研究中，发现响应于斑马鱼幼虫中病原体相关分子模式的新基因也响应于成年斑马鱼和小鼠中的感染刺激，强调了这种新的先天免疫模型用于基因发现的实用性。据推测，斑马鱼幼虫感染刺激的转录反应将揭示新的基因介导的先天免疫哺乳动物。为了检验这一假设，将实现两个目的，1）将采用新的斑马鱼幼虫测定法来确定在不存在适应性免疫的情况下对病原体刺激的全生物体转录应答，以及2）将在斑马鱼和哺乳动物先天免疫应答中验证功能上未表征的病原体应答基因的作用。拟议研究的长期目标是在整个生物体的背景下，在多个尺度上，从个体基因到转录组，开发先天免疫反应的综合模型。成功完成所提出的实验将促进新的未来研究，包括透明斑马鱼幼虫中感染反应的体内可视化，基于全生物体的化学遗传和诱变筛选以鉴定免疫应答的药理学和遗传修饰剂，以及基因敲除/敲低研究以评估这些免疫应答基因在斑马鱼和哺乳动物的免疫防御和恢复中的作用。这些数据、试剂和工具将构成用于研究、评估和调节脊椎动物先天免疫应答的新的基于系统的多生物体范例。 公共卫生相关性脊椎动物在生物体水平上对感染作出反应的完整动态过程尚不清楚。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有反应的未表征的哺乳动物基因。通过这些方法产生的数据将为基于生物体的感染基因组反应提供前所未有的见解，促进用于调节免疫反应的新型诊断，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.