PT Unknown AU Saphörster, J TI Molecular mechanisms of cellular immune responses in marine invertebrates PY 2013 PU Christian-Albrechts-Universität zu Kiel WP https://macau.uni-kiel.de/receive/diss_mods_00011813 LA en DE Transcriptome; bivalves; Mytilus edulis; Lophotrochozoa; evolution; immune system; hemocytes; ROS production; NOX; DUOX; IL-17 AB Invertebrate and vertebrate organisms alike rely on efficient immune mechanisms to cope with potentially harmful microbes. Immune cells of invertebrates, e.g. hemocytes in bivalves, perform innate but not adaptive immune responses as found in lymphatic cells of jawed vertebrates. Since several genes and principles of innate immunity are highly conserved throughout the animal kingdom, invertebrates are sometimes even regarded as a simplified model of the vertebrate innate immune system. Most invertebrate animals belong to one of the three major bilaterian clades: Deuterostomia, Ecdysozoa or Lophotrochozoa. Whereas research on innate immunity was mainly conducted in the first two groups, information about molecular immune mechanisms in the Lophotrochozoa, including molluscs such as bivalves, is still scarce. In addition, marine, invertebrate filter feeders, such as bivalves, are especially suited to elucidate evolutionary conserved immune responses, since they are constantly in direct contact with the surrounding sea water and, hence, with the microbiota within. Therefore, this project aimed to identify and characterize putative conserved and important proteins and pathways of innate immune responses at molecular level in bivalves, including the ocean quahog, Arctica islandica, the Antarctic bivalve Laternula elliptica and especially the ecologically and economically important blue mussel Mytilus edulis. To provide the molecular basis for subsequent investigations, transcriptome datasets of unstimulated and flagellin-challenged M. edulis hemocytes were generated using massively parallel sequencing techniques (Roche 454). Comparative analysis of putative conserved pathways in the generated and existing M. edulis transcriptome datasets displayed an enrichment of putative immune relevant pathways in hemocytes. Furthermore, deeper insights into the time-dependent mRNA expression pattern of selected receptors, regulators and effectors of potential immune pathways in M. edulis revealed early- (e.g. NF-κB pathway members) and late-inducible (e.g. Toll-like receptors) immune gene orthologs in response to flagellin stimulation. The transcriptome datasets also offered the molecular background to detect and characterize phylogenetic ancient immune gene orthologs in bivalves possessing also a great relevance in the human immune system, such as genes potentially coding for NOX/DUOX enzymes and IL-17-like cytokines. One important and phylogenetic conserved immune response investigated in this study was the production of reactive oxygen species (ROS). In humans and also lower animals, such as sea urchins and nematodes, enzymes of the NADPH oxidase (NOX) and dual oxidase (DUOX) family are mainly responsible for the production of ROS. Accordant to various other studies, hemocytes of M. edulis depicted a clear dose- and time-dependent ROS production in response to immune stimulation in the present study. But despite the large number of studies dealing with the generation of ROS in molluscs, the present study was the first to identify putative NOX and DUOX orthologs as well as their subunits in bivalves and even presents one of the first studies in the whole superphylum of Lophotrochozoa. Phylogenetic analyses of these orthologs depicted a closer relationship of deuterostome DUOX orthologs to Lophotrochozoan than to Ecdysozoan orthologs, which underlines the suitability of Lophotrochozoan model organisms for the investigation of the innate immune system from an evolutionary perspective. Furthermore, DUOX orthologs clustered in three distinct groups in the phylogenetic analysis, which mostly coincide with their specific expression pattern among tissues. Orthologs comprising the DUOX-b cluster displayed a particularly high mRNA expression in hemocytes in contrast to other tissues and even to other major immune gene orthologs. On the other hand, the fast and high induction of DUOX expression upon immune stimulation did not correlate with ROS production in M. edulis hemocytes. Nevertheless, the high transcript levels of DUOX-b in hemocytes, its increased transcription in response to immune challenge and a reduced ROS production, when using the inhibitor BAPTA, provides strong evidence for a role of DUOX-b in the innate immune response of bivalves. Mammals use a multitude of cytokine families to communicate within and between cells. Many of these families cannot be found in invertebrates; one major exception are proteins belonging to the interleukin 17 (IL 17) family of cytokines. In the present study, transcripts of two potential IL-17 orthologs were identified and characterized in M. edulis and L. elliptica as well as one ortholog in A. islandica. At least one ortholog in each bivalve depicted a high mRNA expression in gill tissue in comparison to other tissues and hemocytes. In hemocytes of M. edulis, however, transcription of both IL-17 orthologs was highly inducible using various immune stimulants. The resulting expression profiles varied depending on the ortholog and stimulus applied, supporting the presence of two distinctive orthologs with potentially differing functions in bivalves. This study provided a deeper understanding of flagellin-mediated defense mechanisms in M. edulis. Additionally, it gave insights into the conservation and transcriptional regulation of two families of immune gene orthologs in a phylogenetic older group of animals. Thereby, the present study contributes to a more complete view on the evolution of the innate immune system itself. PI Kiel ER