The Ancient Innate Immune System in Sea Urchins

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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.