STUDIO DEL TESSUTO LINFOIDE ASSOCIATO ALL’INTESTINO (GALT) PER APPLICAZIONI BIOTECNOLOGICHE IN ACQUICOLTURA DI SPECIE ITTICHE

The mucosal surfaces represent a major interface between the organism and the external environment. Although evidence has accumulated for the existence of mucosal immunity mechanisms in fish, as it would be expected for animals living in a pathogen-rich aquatic environment, this field of research is still poorly explored. The mucosae of fish skin, gills and gut are protected by both humoral and cellular mechanisms (Peleteiro & Richards, 1985; Hart et al., 1988; Rowley et al., 1988; Roberts, 1989). Leucocytes occur in all parts of the teleost digestive system, most extensively in the intestine, where lymphocytes, plasma cells, granulocytes and macrophages are present in and under the epithelium. Although large lymphoid centres are lacking, many lymphoid cells, either scattered or in small groups, were reported to be present in the epithelium and lamina propria (Zapata, 1979; Davina et al., 1980; Temkin & McMillan, 1986; Hart et al., 1988). Various studies indicated that regional differences occur in the organisation of the gut-associated lymphoid tissue (GALT) at different levels of the digestive tract, possibly reflecting specialised immunological functions. Uptake and transport of antigens have been shown to occur mainly in the second gut segment of carp, Cyprinus carpio L. (Rombout et al., 1985, 1986; Rombout & Van den Berg, 1989), and trout, Oncorhynchus mykiss (Georgopoulou et al., 1986), where antigens are transported from the lumen to large intraepithelial macrophages, which can exert an antigen-presenting function (Rombout et al., 1985, 1986). These findings demonstrated the high immunological relevance of the fish GALT. The study of GALT cellular components, their specific location, morphological and functional characteristics is crucial to understand the role of GALT within the whole fish immune system. The main objective of this thesis was to improve the knowledge about the cell-mediated intestinal immunity in two fish species of great economic importance: the European sea bass (Dicentrarchus labrax L.) and the common carp (Cyprinus carpio L.). Special attention has been devoted to cell-mediated cytotoxic response, among defence mechanisms against foreign (allogeneic or xenogeneic) or altered (e.g. virus-infected) cells. In higher vertebrates, cell-mediated cytotoxicity (CMC) is exerted by NK cells or CTLs, respectively, as the cellular parts of the innate or the adaptive immune defence. NK cell killing is triggered by invariant receptors that recognize molecular patterns on infected cell surfaces. CTLs specifically recognize and kill cells that present peptides derived from antigens synthesized intracellularly (e.g. viral proteins) with major histocompatibility complex (MHC) class I molecules on the cell surface. Recognition of the MHC/peptide complex is mediated by the T cell receptor (TCR). During the last years, MHC class I-restricted effector/target cell recognition systems have been established in some fish species, but the abovementioned mechanisms have been shown in principle but not fully proven. A main problem is that cells performing CMC in fish are still poorly characterized due to the lack of appropriate tools for the specific recognition of lymphocyte populations. Cell-specific markers are indispensable for cell identification, especially of T lymphocyte subpopulations, which seem to have a key role in this type of response. The panel of available monoclonal antibodies (mAb) to fish leucocytes is still very limited: an anti-thymocyte mAb (DLT15) reacting with peripheral T cell populations of sea bass (Scapigliati et al., 1995), a mAb (WCL9) reacting with early T cells of carp (Rombout et al., 1997), a mAb against carp intestinal T cells (WCL38) (Rombout et al., 1998) and a mAb reacting with a T cell population in channel catfish (Passer et al., 1996). In addition, a mAb (5C6) raised against channel catfish, Ictalurus punctatus, non-specific cytotoxic cells (NCC; Ig- lymphoid cells), apparently reacting with NK cells in a variety of vertebrate species (Evans et al., 1988; Evans and Jaso-Friedman, 1992). The existence of anti-T cells appropriate MAbs in carp and sea bass has been a main reason to study here these two fish species. Moreover, homologous sequences of TcRβ, CD8α, CD4 and MHCII-β, four important genes involved in T cell function, became recently available for sea bass. These sequences were used to perform quantitative RT-PCRs and in situ hybridization studies.The results obtained with these molecular techniques, together with those obtained by the use of cell-specific mAbs (FACS analysis and immunohistochemistry), suggested that in sea bass and carp GALT mainly consists of T cells and likely exerts cytotoxic activity (Rombout et al., 1998; Guerra, PhD Tesis 2008). The aim of this thesis was to confirm these hypotheses with functional tests. In fish, allogeneic and xenogeneic cells represent standard model targets of cytotoxic effector cells.Specific and non-specific cytotoxicity assays and different immunisation protocols were developed for the carp and the sea bass using a Epithelioma papulosum cyprini (EPC) carp cell line, a continuous embryonic cell line from European sea bass (DLEC) and a human erythroleukemic cell line (K562) as target cells, and the release of lactate dehydrogenase (LDH) as read out system (Cytotox 96® kit PROMEGA).Non-specific cytotoxicity against EPC was not observed in non-immune specimens, while carp immunised i.p. with EPC clearly showed specific killing by effector cells [purified lymphocytes (90-95% by FACS) by discontinuous Percoll gradient, density 1,02-1,06] present in kidney, spleen and blood, but not in the gut. These results agree with a previous study (Companjen et al.,2006). Otherwise, non-specific cytotoxicity against K562 cells (xenogeneic) was observed in non-immune specimens, ascribed to purified lymphocytes from head kidney, spleen and PBL (negligible in lymphocytes from the intestine). Immunisation with K562 evoked a net increase in cytotoxic responses against the K562 cells, compared with PBS treated controls. The effect was registered in systemic organs (head kidney and spleen), but especially in the intestine. The anal immunisation protocol with lysed cells was the most effective in increasing the CMC against xenogeneic target cells of both systemic and mucosal organs. Taken together, these data can lead to various conclusions: 1) carp apparently lacks non-specific allogeneic cytotoxicity, 2) non-specific xenogeneic cytotoxicity is only systemic (e.g. in head kidney, spleen and PBL), 3) specific cytotoxicity can be induced in kidney and spleen cells following i.p. immunisation with allogeneic cells (EPC), 4) cell-mediated cytotoxic response against xenogeneic cells (K562) can be increased in the intestine and systemic organs by immunisation (anal or i.p).In the sea bass, GALT immunohistochemical analysis preceded the study of CMC. For this study, a polyclonal antibody (A0452 DAKO) raised in rabbit against huma n CD3 ε was used. This pAb has been shown to react wi t h blo o d leuco c y t e s in snapp e r, Pagrus auratus (Coo k et al., 2001), and Japanese flo u n d e r, Paralichthys olivaceus (Park et al. 2005). Th is antib o d y also detects CD3 ε in vari o u s mam ma l s (Jones et al., 1993) and the amp hibian Xenopus laevis (Göbel et al. 2000), illu strating the conser vation of the epito p e. The demonstration of reactivity of this antibody towards CD3 ε in snap per and flounder has identified a candidate antibody for the detection of T cells in other teleost species. Th is antib o d y showed cross-reactivity with lymphocytes of sea bass. The localization and distribution of CD3+ cells in the sea bass intestinal mucosa are here shown for the first time. The comparison between the data obtained with the mAb DLT15 and those obtained with the anti-CD3 ε pA b showed that the majority of sea bass intra-epithelial lymphocytes (IEL) is a CD3ε – population. Otherwise, most IEL of the carp seem to be CD3ε +. Further studies are needed to assess the putative occurrence of T cell subpopulations in the intestinal mucosa , and to explain the observed species differences.In sea bass, lymphocytes isolated from head kidney, spleen and gut exhibited non-specific cytotoxicity against DLEC (allogeneic cell line). The highest activity was observed in intestinal lymphocytes, although significant responses were also seen in spleen and head kidney lymphocytes. Also the non-specific cytotoxicity against K562 (xenogeneic cell line) was significant in lymphocytes purified from head kidney, spleen, blood, gut and gills. These results suggest a wide occurrence of xeno-reactive cells in sea bass organs, that in carp were localized only in systemic compartments. Following anal immunisation with K562, cell-mediated citotoxicity against xenogeneic targets decreased significantly both in systemic and mucosal organs, compared with controls. Therefore it can be hypothesised that this decrease may be due to induction of tolerance, but further studies are needed to test this hypothesis. Such informations would improve the efficacy of oral vaccination in sea bass and optimize the antigen delivery to the appropriate cellular targets. A very promising procedure to improve the mucosal immune defence mechanisms is the dietary administration of probiotics, that appeared as a effective tool for the control of pathogen outbreaks in fish farms. Many authors have repo r te d increase d disease resista n ce in fish fed bacter ia-supplemented diets (Bly et al., 1997; Robertson et al., 2000; Spanggaar d et al., 2001; Raid a et al., 2003). In this thesis we investigated the effects of the oral administration of heat-inactivated Lactobacillus delbrüeckii ssp. lactis and Bacillus subtilis, individually or combined, on immune responses, both systemically and locally in the gut, in a further marine teleost species, the gilthead seabream (Sparus aurata L.). In a first experiment, seabream (65 g) were fed for 3 weeks different diets supplemented with 1 x 107 CFU g-1 Lactobacillus, 1 x 107 CFU g-1 Bacillus, or 0.5 x 107 CFU g-1 Lactobacillus plus 0.5 x 107 CFU g-1 Bacillus. Controls were fed non-supplemented diet. Six fish per group were sampled at the end of the trial and some humoral and cellular systemic innate immune parameters were evaluated. Feeding the mixture of the two killed bacteria species significantly increased natural complement , serum peroxidase and phagocytic activities compared with controls. In a second experiment, juvenile seabream (13 g) were fed for 3 weeks the same experimental diets and total serum IgM and numbers of gut Ig M + cells and acido philic granulocytes were evaluated. All these parameters were significantly higher in the multispecies probiotic group compared to monospecies and control fed groups. The results of the present study indicated that oral administration of heat-inactivated probiotics has stimulatory effects, both local and systemic , on the immune system of the gilthead seabream. The multispecies formulation was more effective than any of the sing le-bacteria experimental diets. These results suggest that, as in humans, the appropriate design of multispecies probiotics, including inactivated cells, can have synergistic positive effects on fish health.