Risposte immunitarie nei vertebrati ectotermi: attività cellulari e molecolari di leucociti del teleosteo Dicentrarchus labrax

Abstract

I pesci ossei si sono originati circa 400 milioni di anni fa e rappresentano il gruppo più numeroso dei Vertebrati viventi con più di 20000 specie conosciute. I Teleostei hanno subito una lunghissima selezione evolutiva che ha consentito loro di colonizzare tutti gli ambienti acquatici presenti sul pianeta, anche i più estremi: dalle profondità degli oceani fino ai fiumi del Tibet, dando luogo ad una straordinaria radiazione adattativa. Abitudini ed ambienti di vita così diversi espongono i pesci ad un’enorme varietà di parassiti e patogeni che esercitano una notevole pressione selettiva sul loro sistema immunitario. Quest’ultimo, grazie alla sua flessibilità, si è specializzato a tal punto da diventare uno dei probabili motivi del grande successo evolutivo dei pesci. Negli ultimi decenni l’itticoltura ha avuto un incremento notevole su scala mondiale e, per far fronte a questa nuova richiesta, è pratica comune negli impianti allevare gli animali in condizioni di sovraffollamento. Esse, unitamente a fattori di stress quali temperature inadeguate, sbalzi di salinità, scarsa qualità dell’acqua, possono facilitare la diffusione rapida di patologie infettive, in particolare durante gli stadi larvali dei pesci. La spigola europea o branzino (Dicentrarchus labrax L.) è una specie marina di grande importanza economica, specie nell’acquacoltura del Mediterraneo. Tuttavia, numerosi virus patogeni, batteri, funghi e parassiti influenzano la specie, causando varie malattie infettive. Tra queste patologie l’encefalopatia virale, la retinopatia (Bovo et al., 1999. Ucko et al, 2004), la pasteurellosi e le vibriosi causate da batteri patogeni come Photobacterium damselae e Vibrio anguillarum e dal virus dell’encefalite, determinano le perdite più pesanti in acquacoltura per la spigola. Negli ultimi decenni lo studio del sistema immunitario delle specie di allevamento è divenuto uno dei principali obiettivi di indagine sperimentale, per le ovvie ricadute fondamentali sulla prevenzione e il trattamento delle patologie e la tutela della salute delle specie allevate. In questo senso, la conoscenza molecolare e i meccanismi genetici alla base della resistenza agli agenti patogeni potrebbero essere di notevole aiuto nello sviluppo di vaccini efficaci e strategie appropriate di vaccinazione (Bricknell and Dalmo, 2005; Chinabut and Puttinaovarat, 2005). In questi ultimi anni, diversi studi hanno chiaramente evidenziato che la tecnica con il più basso fattore di stress per gli individui è quella della vaccinazione per immersione. Tuttavia, ad oggi, poche sono le informazioni relative all’immunità di tipo mucosale nei pesci ossei. In questo contesto diventa fondamentale lo studio dell'immunità e in particolare delle mucose quali branchie, pelle ed intestino che sono i siti esposti al contatto diretto con gli antigeni esterni e quindi potenziali target/veicoli per i processi di immunizzazione. La ricerca di base che tenta di comprendere i meccanismi di funzionamento del sistema immunitario dei Teleostei trova spazio proprio in tale contesto. Negli ultimi venti anni, l’attenzione della ricerca si è rivolta verso l’identificazione di marcatori immunitari specifici, necessari per una conoscenza dettagliata dei meccanismi molecolari e delle popolazioni cellulari primariamente coinvolte nelle risposte immunitarie. Recentemente sono stati clonati nei teleostei molti dei principali geni che codificano per molecole immunomodulatorie coinvolte nelle risposte immunitarie innate ed acquisite, omologhe a quelle dei Mammiferi (Scapigliati et al., 2006; Randelli et al., 2008). Nonostante questo rapido progresso, le conoscenze sull’immunologia funzionale nei pesci teleostei sono scarse, soprattutto per la carenza di marcatori specifici per cellule e molecole coinvolte nei processi immunitari. Il presente progetto di dottorato di ricerca si è principalmente rivolto ad ampliare le conoscenze relative al sistema immunitario della spigola o branzino (Dicentrarchus labrax L.). In questa specie, particolare interesse ha rivestito lo studio delle popolazioni linfocitarie residenti negli organi mucosali, delle quali si è valutato attraverso test biologici e funzionali l’attività proliferativa in risposta a particolari stimolazioni mitogene. In aggiunta, parte di questo lavoro ha previsto di valutare anche la funzione e il ruolo, nella risposta immunitaria, di un importante marcatore linfocitario, il corecettore CD45. Una maggiore conoscenza dei meccanismi alla base dell’attivazione linfocitaria risulta indispensabile per comprendere ed ottimizzare, nei pesci, le tecniche di immunostimolazione nella vaccinazione orale.

Aquaculture, the farming of aquatic organisms, has been the biotechnology activity with the highest growth rate worldwide in the last four decades. The annual aquaculture production is at present over 60 million tons, with an approximate value of 85 billion dollars (FAO, World Review of Fisheries and Aquaculture, 2010). Despite the undeniable benefits of aquaculture such as the provision of good quality and accessible food for population and the generation of millions of jobs and billion dollars in budget for the developing countries, the activity is one of the most criticized worldwide, mainly because of the environmental impacts that have been and can be caused. Moreover, under intensive culture conditions, fish are subject to increased stress owing to environmental (water quality and hypoxia) and health conditions (parasites and infectious diseases). All these factors have negative impacts on fish well-being and overall performance, with consequent economic losses (Teles, 2012). European sea bass (Dicentrarchus labrax L.; Moronidae; Perciformes) is a marine species of great economic importance, especially in Mediterranean aquaculture. However, numerous pathogenic viruses, bacteria, fungi and parasites affect the species, causing various infectious diseases. Among those pathologies, viral encephalopathy and retinopathy (Bovo et al., 1999; Ucko et al., 2004), pasteurellosis and vibriosis (Afonso et al., 2005) caused by D. labrax encephalitis virus and bacterial pathogens like Photobacterium damselae subsp. piscicida and Vibrio anguillarum, respectively, lead to the most heavy losses in aquaculture production of sea bass. In this respect, knowledge on molecular and genetic mechanisms of resistance to pathogens and specific features of immune response against various infectious agents should greatly benefit the development of effective vaccines and proper vaccination strategies in markerassisted selection (MAS) of fish resistant to a range of infections (Bricknell and Dalmo, 2005; Chinabut and Puttinaovarat, 2005). During the last decades vaccination has become established as an important method for prevention of infectious diseases in farmed fish. The majority of aquatic vaccines are delivered by injection, which is by far the most effective method when compared to oral or immersion deliveries. However it is labour intensive, costly and not feasible for large numbers of fish. Attempts to develop novel oral and immersion delivery methods have resulted in varying degrees of success but may have great potential for the future (Plant and Lapatra, 2011). Recent data have shown that immune-stimulation and vaccination may modulate transcriptional levels of CD8α and/or CD4 in some fish species and, together with data showing the antigen uptake occurring in gills and in the intestine, it appears evident that studies on T cells subtypes and their distribution and modulation is important to develop strategies for oral and immersion vaccination. As physical barriers that separate teleost fish from the external environment, mucosae are also active immunological sites that protect them against exposure to microbes and stressors. In mammals, the sites where antigens are sampled from mucosal surfaces and where stimulation of naive T and B lymphocytes occurs are known as inductive sites and are constituted by mucosa associated lymphoid tissue (MALT). According to anatomical location, the MALT in teleost fish is subdivided into gutassociated lymphoid tissue (GALT), skin-associated lymphoid tissue (SALT), and gillassociated lymphoid tissue (GIALT). All MALT contain a variety of leukocytes including, but not limited to, T cells, B cells, plasma cells, macrophages and granulocytes. In this view, the main objective of this thesis was to improve the knowledge about mucosal immunity in the European sea bass. Special attention has been devoted to study mechanisms of cellular in vitro proliferation of gills and gut leucocytes after mitogen stimulation, in order to provide experimental evidence on leukocyte populations involved and on their mechanisms of action in mucosal immunity. To date, some specific cellular markers (anti-sea bass T cell marker DLT15/DLT22, anti-sea bass B cells marker DLIg3) and molecular markers (TCR α-β -γ, CD8α, CD4, TNF-α, IL-1β, MHC-II, IFN-α, IgM, RAG-1, COX-2, MX, etc.) are available in sea bass. The first step it was to set up a method for studying cellular proliferation of leucocytes of sea bass, and a number of experiments has been made in order to develop optimal conditions of cell growth. Cells obtained from different organs (spleen, intestine, gills, head kidney) and enriched in leucocytes by Percoll with discontinuous gradient density 1.02 g/cm3 and 1.07 g/cm3, were stimulated with two different lectins, such as PHA (phytohemagglutinin) and ConA. (concanavalin A). The cells proliferation was then measured by the fluorescence-based CFSE assay and monitored by flow cytometry. This type of study is rather innovative, because in literature there are few information about in vitro mechanisms of leukocyte proliferation in fish after mitogen stimulation. In the next step, the target cells for the study were taken from gills and gut tissues. The data obtained clearly show that a lectin-induced proliferation of gills and gut leucocytes, and that this proliferation was associated with an increase of DLT15 and DLT22-labelled cells. At last, we have measured the trascription level of T genes from 12 individuals after 24h and 48h stimulation with PHA and ConA. Also, part of my work has been focused on T cells population and mechanism of lymphocyte activation mediated by co-receptors such as CD45. In mammals, the cell surface-associated CD45 receptor molecule is a highly glycosylated and high molecular weight enzyme tyrosine phosphatase (Tonks, 1988), also known as leukocyte common antigen, a type I transmembrane protein present on all hemopoietic cells, except erythrocytes (Thomas, 1989; Trowbridge, 1991; Trowbridge and Thomas, 1994). The CD45 is a fundamental regulator of B- and T-cell antigen receptor signalling (Altin and Sloan, 1997): its long cytoplasmic domain transduces the extracellular signal through a phosphatase activity that in T cells activate Lck tyrosine kinases, or Lyn/Fyn/Lck kinases in B cells (McNeill et al., 2007). This co-receptor therefore seems to have a role in the activation of T lymphocytes and in the immune response. The CD45 receptor gene can be expressed in mammals leucocytes in five alternatively spliced glycoproteins differing in the extracellular domain, and regulated in various cell-type specific isoforms that differ in size, shape and negative charge (Penninger, 2001), with the precise role of these isoforms still remaining elusive (Falahati and Leitenberg 2007). Naïve T lymphocytes express high molecular weight CD45 isoforms (around 220 kDa in size), and are usually positive for CD45RA, whereas after thymic maturation activated and memory T lymphocytes express CD45RO, a shorter CD45 isoform at 180 kDa (Hathcock et al., 1992). The central memory CD45RO T helper cells are CCR7+ and CD62L+, whereas effector memory cells are CCR7- and CD62L- (Sallusto et al., 2004; Krakauer et al., 2006). The biological significance of the various isoforms of the CD45 gene expressed in leucocytes is still matter of debate (McNeill et al., 2007, Kozieradzki et al., 1997; Dawes et al., 2006; Earl and Baum, 2008), but it is assumed that individual isoforms may play important roles in lymphocyte activation and development. In order to better investigate some features of immune-biology of CD45 in vertebrates, we employed the European sea bass as a fish model, and in this species we investigated the transcription of the CD45 gene under in vitro stimulation of leucocytes. By obtaining an anti-CD45 mAb, called DLT22, we also studied the distribution of CD45-bearing leucocytes in lymphoid tissues of sea bass, and the involvement of CD45-bearing cells during lectin-induced proliferation of leucocytes. We observed a marked difference in the percentages of staining, being very high in thymocytes (>90%), intestinal leucocytes (>80%), and gill leucocytes (>75%), and very low in head kidney, spleen, and PBL (around 1-4 % for each tissue). To obtain information on the molecular size of the DLT22 antigen, western blotting experiments were performed on cell lysates from thymus and has been observed the presence of multiple immune-reactive bands. The 130 kDa band was excised from the gel and subjected to sequence analysis, and when obtained product were compared to a fish peptide database, two peptide sequences matched with CD45 of Takifugu rubripes. The selected sequence RYVDILPYDYNRV, matched at position 649-659 of channel catfish CD45, corresponding in this latter species to enzymatic PTPase domain I. From these data we strongly supposed that the mAb DLT22 recognized as antigen an epitope present on sea bass CD45 molecule. In this respect, we have decided to clone partially the CD45 sea bass gene in order to perform various functional tests described in detail later in this thesis work.