T cell transcripts and T cell activities in the gills of the teleost fish sea bass (Dicentrarchus labrax)
Introduction
The gills of fish are, in terms of exposed surface, the biggest tissue of most Teleost species (e.g. 1 m2/kg in carp; Oikawa and Itazawa, 1985), they serve to maintain fish omeostasis by the uptake of nutrients and substances, and by forming an active barrier against the entry of pathogens. The thin gill epithelium is a mucosal tissue at direct contact with the water environment, and contains a gill-associated lymphoid tissue (GIALT) with macrophages/granulocytes (Barnett et al, 1996, Lin et al, 1998, Mulero et al, 2008), B cells (Davidson et al, 1997, Dos Santos et al, 2001, Salinas et al, 2011), T cells (Scapigliati et al., 1999), and with high expression levels of T cell-related genes (Boschi et al., 2011). The GIALT is a first-line of defense against the entry of pathogens from the microbe-rich water environment and thus must be armed with either a fast non-self recognition and elimination system, together with an antigen recognition/antigen memory asset.
For these features of innate and acquired immune defenses, the GIALT is regarded as a target tissue for mucosal vaccination and, effectively, vaccination of some fish species is achieved by brief immersion of fish in antigenic mixtures diluted in water, from which the antigen enters the animal through the gills and other mucosal tissues (Salinas et al., 2011).
Of particular importance are lymphocytes of mucosal tissues, where T cells may be present in percentages up to 60% in gut-associated lymphoid tissue (GALT) and of 25% in GIALT (Randelli et al., 2008) and, as in mammals, are considered to be involved in a first line of defense against pathogen entry. However, despite the obvious importance of GIALT in maintaining fish health, the knowledge on its cellular and molecular components is still meagre.
In order to extend our previous observations on T cells and T cell transcripts (Boschi et al, 2011, Pallavicini et al, 2010, Randelli et al, 2009), and to achieve a more comprehensive knowledge of GIALT asset and function in fish we employed the European sea bass (Dicentrarchus labrax) as a model to produce a whole gill RNA transcriptome and analyze it for the presence of genes coding for T lymphocyte-related peptides. Moreover, we investigated some basical cellular aspects typical of T cells like in vitro induction of proliferation by lectins and expression of T cell-related genes during proliferation, an argument poorly known in fish.
Section snippets
Fish and leucocytes
Healthy juveniles of sea bass were obtained from a local fish farm (Civitaittica SrL, Civitavecchia, Italy). The fish were lethally anesthetized with tricaine methanesulfonate (Sigma) and blood was drawn from caudal vein with a syringe. The gills were then removed and immersed in cold HBSS, branchial arches were gently cleaned with filter paper to remove external mucus and gill filaments cutted out with small scissors in a Petri dish. The fragments were finely minced in HBSS with the aid of a
Transcriptome sequencing
The Illumina sequencing of Dicentrarchus labrax gills transcriptome produced 68,643,808 raw nucleotide paired-end reads. The average read length was 100 bp, corresponding to a complete dataset of 6.86 GB of sequence data. This set was reduced to 6.56 GB following the trimming procedure, which removed short reads and low quality or ambiguous nucleotides (the average reads length was thus reduced to 98.7 bp). Trimming statistics are reported in Appendix: Supplementary Table S1a. The estimated
Discussion
The gills of fish have an obvious importance for the maintaining of homeostasis by regulating exchanges with the outside water microbiome, but despite this fundamental feature the knowledge on cellular and molecular asset of mucosal immune system in this tissue is limited. Considering the importance of T lymphocytes in mucosal immune defenses of vertebrates, the aim of our work was to investigate basic molecular and cellular signatures of T cells in the gills of sea bass by transcriptome
Acknowledgements
This research was funded by the European Commission under the 7th Framework Programme for Research and Technological Development (FP7) of the European Union (Grant Agreement 311993 TARGETFISH).
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These authors contributed equally to the work.