T cell transcripts and T cell activities in the gills of the teleost fish sea bass (Dicentrarchus labrax)

https://doi.org/10.1016/j.dci.2014.07.015Get rights and content

Highlights

  • A de novo transcriptome from unstimulated gills of sea bass has been obtained.

  • The gills of sea bass express a set of T cell subpopulations-related genes similar to that known in mammals.

  • T cells of gills proliferate in vitro in response to lectins.

Abstract

The gills of fish are a mucosal tissue that contains T cells involved in the recognition of non-self and pathogens, and in this work we describe some features of gill-associated T cells of European sea bass, a marine model species. A whole transcriptome was obtained by deep sequencing of RNA from unstimulated gills that has been analyzed for the presence of T cell-related transcripts. Of the putative expressed sequences identified in the transcriptome, around 30 were related to main functions related to T cells including Th1/Th2/Th17/Treg cell subpopulations, thus suggesting their possible presence in the branchial epithelium. The number of T cells in the gills of sea bass, measured with the specific T cell mAb DLT15 range from 10% to 20%, and IHC analysis shows their abundance and distribution in the epithelium. Leukocytes from gills are able to proliferate in the presence of lectins ConA and PHA, as measured by flow cytometry using CFSE fluorescence incorporation, and during proliferation the number of T cells counted by immunofluorescence increased. In lectin-proliferating cells the expression of T cell-related genes TRβ, TRγ, CD4, CD8α, CD45 and IL-10 increased dramatically. Our data represent a first analysis on T cell genes and on basic T cell activities of fish gills, and suggest the presence of functionally active subpopulations of T lymphocytes in this tissue.

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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