The Ancient Innate Immune System in Sea Urchins

该项目的目的是广泛了解紫色海胆Strongylocentrotus purpuratus的古老先天免疫系统，这是一种在缺乏适应机制的情况下发挥作用的防御系统。在海胆中鉴定并部分表征了一种更简单的替代补体系统，其由SpC3和SpBf组成，SpC3和SpBf分别是脊椎动物C3和因子B（Bf）的同源物。虽然这种更简单的补体系统似乎是重要的宿主防御在这种动物中，其他组件预计将存在。我们提出了两种方法来识别海胆免疫系统的其他组成部分。首先，我们将构建一个标准化的，扣除的cDNA文库从激活的体腔细胞（免疫细胞）的基础上扣除使用的信息从免疫静止体腔细胞。免疫静止细胞将从海胆获得，在体腔液或体腔细胞中没有可检测的SpC3，即，这些动物由于被圈养在我们高质量的封闭海水系统中而降低了免疫系统。对来自该文库的克隆的分析将导致鉴定在活化的体腔细胞中特异性表达的基因，其中许多将是免疫应答基因。其次，我们将使用最近克隆的海胆同源物的转录因子核因子κ B（NF κ B），称为SpNF κ B，以确定额外的免疫反应基因。哺乳动物和昆虫的先天免疫在很大程度上受转录因子NFkB或Rel家族成员的控制。我们将分离并测序几个免疫应答基因的启动子，我们知道这些基因是通过免疫攻击上调的（SpC3，SpBf，SpNFkB和凝集素Sp056），以确定结合NFkB蛋白的共有位点，称为kB位点。这将启动未来的研究，通过比较这些启动子上的共识结合位点，并通过凝胶迁移试验控制海胆免疫反应的监管系统。我们还将使用抗SpNFkB抗血清进行染色质免疫沉淀，以分离与kB位点相关的基因，并可能由SpNFkB控制。这将构成包括免疫反应基因的第二组基因。这些努力的结果将对我们对先天免疫的整体理解产生许多影响。我们的知识的免疫功能在一个简单的后口将显着扩大。基于棘皮动物（包括海胆）和脊索动物（包括哺乳动物）之间的系统发育关系，这些结果也将扩大我们对高等脊椎动物复杂的先天免疫系统的理解，并将阐明宿主防御的核心和必要方面。(The海胆补体系统是对应于哺乳动物系统中的基本和更复杂组分的组分的实例。最后，结果将提供一个很好的估计免疫系统的功能在祖先的有颌脊椎动物的时候，祖先的RAG基因入侵的基因组中的逆转录转座子。这一事件为脊椎动物祖先引入了基因重排能力，这是适应性免疫系统进化所必需的。对海胆中祖先先天免疫系统的分析将使我们能够预测脊椎动物祖先中存在的防御系统的类型，该防御系统负责RAG样基因的选择和维护，以允许适应性免疫系统的进化发生。

The aim of this project is a broad understanding of the ancient innate immune system of the purple sea urchin Strongylocentrotus purpuratus, a defense system that functions in the absence of adaptive mechanisms. A simpler alternative complement system has been identified and partially characterized in the sea urchin which is composed of SpC3 and SpBf, homologues of vertebrate C3 and Factor B (Bf) respectively. Although this simpler complement system appears to be important for host defense in this animal, additional components are predicted to be present. We propose two approaches to identify additional components of the sea urchin immune system. First, we will construct a normalized, subtracted cDNA library from activated coelomocytes (immune cells) based on subtractions using messages from immunoquiescent coelomocytes. Immunoquiescent cells will be obtained from sea urchins with no detectable SpC3 in the coelomic fluid or in the coelomocytes, i.e., animals that have down-regulated their immune system as a result of being housed in our high quality, closed sea water system. Analysis of clones from this library will result in the identification of genes that are specifically expressed in activated coelomocytes, many of which will be immune response genes. Second, we will use the recently cloned sea urchin homologue of the transcription factor Nuclear Factor kappa B (NFkB), called SpNFkB, to identify additional immune response genes. Innate immunity in both mammals and insects is controlled to a great extent by members of the NFkB or Rel family of transcription factors. We will isolate and sequence the promoters of several immune response genes that we know are up-regulated by immune challenge (SpC3, SpBf, SpNFkB, and a lectin, Sp056), to identify consensus sites, called kB sites, that bind NFkB proteins. This will initiate future studies of the regulatory system that controls sea urchin immune responses through comparisons of consensus binding sites on these promoters and by gel shift assays. We will also use anti-SpNFkB antiserum for chromatin immunoprecipitation to isolate genes linked to kB sites, and perhaps controlled by SpNFkB. This will constitute a second set of genes that will include immune response genes. Results from these efforts will have a number of impacts on our overall understanding of innate immunity. Our knowledge of the immune functions in a simple deuterostome will be significantly expanded. Based on the phylogenetic relationship between the echinoderms (including sea urchins) and chordates (including mammals), the results will also expand our understanding of the complex innate immune system of higher vertebrates and will elucidate aspects that are central and essential for host defense. (The sea urchin complement system is the example of a component that corresponds to an essential and more complex component in the mammalian system.) Finally, results will provide a good estimator of the immune system that functioned in the ancestor of jawed vertebrates at the time when the progenitor of the RAG genes invaded the genome in a retrotransposon. This event introduced gene-rearranging capabilities into the vertebrate ancestor that was required for the evolution of the adaptive immune system. Analysis of the ancestral innate immune system in the sea urchin will allow us to predict the type of defense system that was present in the vertebrate ancestor and that was responsible for RAG-like gene selection and maintenance to allow the evolution of the adaptive immune system to occur.