Studi di immunologia funzionale in spigola (Dicentrarchus labrax L.): immunoglobine di tipo T e interleuchina -4/13

Dottorato di ricerca in Genetica e biologia cellulareTeleost fish form a large group with about 20,000 identified species, in comparison to about 10,000 species for birds, and only around 5700 species for mammals. They are adapted to different environments as, for instance, Perciformes that are present both in freshwater and marine habitats, including Antarctic. Fish can be infected by viruses (rhabdoviruses, bornaviruses, reoviruses, nodaviruses, iridoviruses, herpesviruses, etc.), bacteria (Vibrio, Aeromonas, Flavobacterium, Yersinia, Lactococcus, Mycobacterium, etc.), and many other Metazoan parasites. Thus, it is expected that a considerable diversity of host/pathogen interactions characterize fish immune defense mechanisms. European sea bass (Dicentrarchus labrax L.) is a marine species of great economic importance, especially in Mediterranean aquaculture. However, numerous pathogenic viruses, bacteria, parasites affect this species, causing various infectious diseases that lead to the most heavy losses in aquaculture. B and T lymphocytes mediate the specific responses of adaptive immunity in jawed vertebrates. One of the classical B cell function is the production of antibodies (or immunoglobulins) to specifically neutralize pathogens. Immunoglobulins (Igs) are critical factors of the adaptive immune system and they have been found in all vertebrates with jaws (gnathostomes) investigated to date (Flajnik and Kasahara, 2010). Different Igs isotypes have been identified in vertebrates; in teleost fish it was recognized, until about ten years ago, only the presence of two Ig isotypes: IgM, a tetrameric molecule highly used to verify the systemic immune-response against pathogens being the most abundant Ig in the serum (Sunyer, 2012), and IgD, a monomeric immunoglobulin whose function still need to be fully characterized (Edholm et al., 2011). In 2005, a new Ig was found at the same time in rainbow trout (Onchorynchus mykiss) and in zebrafish (Danio rerio) and named in two different ways: IgT (from trout, Hansen et al., 2005) and IgZ (from zebrafish, Danilova et al., 2005), respectively. After that, other IgT/IgZ sequences have been identified in different fish species. The functional characterization of IgT has been first performed in rainbow trout and this molecule has been considered mainly involved in mucosal immunity (Zhang et al, 2010). The first part of this PhD thesis has been focused on sea bass IgT. Several IgT partial sequences were identified from our available sea bass gills transcriptome and allowed to design diverse primers for initial cloning and successive 3’- and 5’-RACE amplification of IgT molecule. The obtained PCR products were used to reconstitute the full-length cDNA corresponding to a sea bass IgT heavy chain subunit (accession number KM410929) composed of 552 aa. The V-domain of sea bass IgT contains the typical amino acids conserved in all sequences of immunoglobulin and T cell receptors (Lefranc 1999 Lefranc et al., 2003). The entire C-DOMAIN could be divided in four CH domains as it happens in most IgT heavy chains. The four sea bass constant domains contain (CH1-CH4) the cysteine and tryptophan residues required for their correct folding (Lesk and Chothia, 1982). A 3D model of sea bass IgT has been generated by comparative modelling after selection of the best template available in PDB. The template structure selected is identified by PDB code 1IGT, an intact Ig gamma-2A monoclonal antibody from mouse. Un-stimulated sea bass juveniles were used to investigate IgT and IgM transcript levels in different organs and tissues. The highest IgT expression was found in muscle, followed by gut, gills and thymus, whereas IgM maximum expression was in brain, followed by spleen, thymus and liver. Moreover, the transcripts levels of IgT in gut and gills were much higher compared to IgM (Figure 1). This suggests that the IgM have a predominant role in systemic responses, whereas IgT is the key participant in mucosal surfaces, as demonstrated in rainbow trout (Zhang et al., 2010). Figure 1. IgT and IgM sea bass basal expression in different tissues. IgT and IgM mRNA levels were expressed as a ratio relative to rRNA 18S levels in the same tissue after real-time PCR analysis using the IgT in brain as calibrator. Data were expressed as the mean ± SD. ** indicates that IgT are significantly different from IgM with p < 0.01; *** indicates that IgT are significantly different from IgM with p < 0.001. ISH analysis of spleen and head kidney sections revealed the presence of IgT-expressing cells in sea bass organs, as demonstrated in mandarin fish (Siniperca chuatsi) (Tian et al.,2009). To investigate IgT localization, we produced a polyclonal antibody against sea bass IgT (raIgT1) and used it to identify the IgT molecule in immunoblotting on total sea bass immunoglobulins purified from head kidney leukocytes. Immunoblot analysis showed that the IgT polyclonal antibody reacted with a band possibly corresponding to sea bass IgT. Under reducing conditions, the molecular mass was of ~75 kDa, in agreement with corresponding results in rainbow trout (Zhang et al., 2010). We used immunofluorescence and immunoistochemistry analysis of spleen and head kidney leukocytes to highlight the IgT+ B and IgM+ B cell subpopulations using raIgT1 and a monoclonal antibody to sea bass IgM (DLIg3). The results confirmed the absence of double-positive IgT+ IgM+ cells and, in this way, we could demonstrate the presence of a sea bass B cell lineage that expressed only IgT, as it happens in rainbow trout (Zhang et al., 2010). The obtained results are of high interest for a preliminary view on the possible function of IgT in sea bass, especially taking into consideration the involvement of the mucosal tissues in the immune response against pathogens. Another subject of this thesis was related to cytokines that interact with T cells. Conventional T cells can be divided into T cytotoxic (Tc) or T helper (Th) cells, distinguished by the expression of the membrane bound glycoproteins CD8 or CD4, respectively. CD4+ T lymphocytes can be further classified according to the expression of specific transcription factors and the secretion of different cytokines combinations in: Th1, Th2, Th17 and Treg. GATA-3, however, is the master transcription factor that mediates the differentiation of Th cells towards a Th2 profile. Mammalian Th2 cells produce the cytokines IL-4, IL-5, and IL-13 that stimulate B cells to secrete different antibody isotypes and thus control extracellular infections. The second part of my PhD thesis was focused on studying the IL4/13 cytokine in sea bass. The first fish IL-4/IL-13 related gene was identified adjacent to RAD50 in the pufferfish (Tetraodon nigroviridis) genome (Li et al., 2007). A second IL-4/IL-13 like gene was discovered in zebrafish (Danio rerio) at a different locus (close to KIF3A) (Bird and Secembes, 2006). Hence, the two genes were called IL-4/13A (close to RAD50) and IL-4/13B (close to KIF3A) (Ohtani et al., 2008). In the sea bass gills transcriptome and sea bass available genome, we have identified two isoforms of the IL-4/13A gene, called IL-4/13A1 and IL-4/13A2 (therefore close to RAD50), and, on another chromosome, we identified a third isoform, called IL-4/13B (close to KIF3A), probably originated by gene duplication events (McCormick and Heller, 2015; Ohtani et al., 2008). IL-4/13 sea bass genes encode proteins of 140 (IL-4/13B), 133 (IL-4/13A1) and 131 (IL-4/13 A2) amino acids, respectively (accession numbers KJ818331-3). Un-stimulated sea bass juveniles were used to investigate IL -4/13 isoforms isoforms transcript levels in different organs and tissues. To gain a further insight into IL-4/13 isoforms involvement in sea bass immune system responses, we performed an in vitro expression analysis by real-time PCR using head kidney and spleen leukocytes stimulated with phytohaemagglutinin (PHA) and phorbol 12-myristate 13-acetate (PMA). PHA stimulation in HK cells increased the expression of IL-4/13B and IL-4/13A1 transcripts after 4 and 24h, whereas in spleen the treatment increased the expression of all IL-4/13 isoforms after 4 h (see table 1), as demonstrated in rainbow trout (Wang et al., 2016). Table 1: PHA stimulation in HK and spleen leukocytes . The data are expressed as high up-modulation of the expression (+++), modest up-modulation (++), light up-modulation (+); as high down-modulation of the expression (---), modest down-modulation (--), light down-modulation (-),* indicates p <0.05. PMA stimulation in HK and spleen leukocytes increased the expression of IL-4/13 isoforms after 4h and IL-4/13B only adter 24 h (see table 2). Table 2: PMA stimulation in HK and spleen leukocytes . The data are expressed as high up-modulation of the expression (+++), modest up-modulation (++), light up-modulation (+); as high down-modulation of the expression (---), modest down-modulation (--), light down-modulation (-), * indicates p <0.05. We can state that, as observed in rainbow trout, both PHA and PMA are able to stimulate the expression of the IL-4/13 isoforms with different kinetics both in head kidney and spleen leukocytes. Moreover, we have produced in E. coli the recombinant sea bass IL-4/13 isoforms and we have analyzed the biological activity of these proteins performing in vitro stimulations on HK and spleen leukocytes. In particular, we have studied the modulation of the expression of different target genes related to the immune system, like cytokine receptors (rIL-4a, rIL-4b, rIL-13a1, rIL-13a2), IL-10, SOCS3, SAP1, hepcidin and different immunoglobulin isotypes (IgM, IgT and IgD). We can state that the recombinant sea bass IL-4/13 isoforms are able to stimulate the expression of the target genes with different kinetics both in the head kidney and in spleen leukocutes. In head kidney cells the recombinant IL-4/13B modulates the expression of target genes after 4h from stimulation, whereas the recombinants IL-4/13A modulate the expression of the majority of target genes after 24h (see Table 3A). In spleen cells the recombinants IL-4/13B and A2 modulate the expression of target genes both at 4 and 24 h after stimulation, while for the recombinant IL-4/13A1 the modulation is less evident (see Table 3B). SAP 1 and HEP are involved in the response to pathogens and have an important role in the activation of the adaptive immune responses. The rapid up-regulation of the expression of SAP1 and hepcidin after 24 h from the stimulation with the IL-4/13A recombinants in HK cells, suggests a role of these type-2 cytokines in the resolution of infection (see also Whang et al.,2016). Rapid up-regulation of IL-4a/b and IL-13a1/a2 expression may suggest a self-amplification mechanism of signaling specific to these cytokines, caused to the specific recognition of the ligand with its receptor. Rapid up-regulation of anti-inflammatory genes, as IL-10 and SOCS3, suggest an anti-inflammatory role of fish type-2 cytokines, as observed in mammals where IL-4 and IL-13 are able to suppress the expression of pro-inflammatory genes (Bhattacharjee et al., 2013). Table 3: Changes of the target gene expression after stimulation with the recombinant IL-4/13 isoforms. A) Modulation of genes in HK leukocytes ; B) Modulation of genes in spleen leukocytes . The data are expressed as high up-modulation of the expression (+++), modest up-modulation (++), light up-modulation (+); as high down-modulation of the expression (---), modest down-modulation (--), light down-modulation (-), empty box means no modulation, * indicates p <0.05 and p<0.01. Finally, we have studied the expression of IL-4/13 isoforms after different vaccination procedures using two pathogens, a virus (the Nodavirus or NNV) and a bacterium (Vibrio anguillarum) (Figure 2). Figure 2: Modulation of the expression of sea bass IL-4/13A1, A2 and IL-4/13B in head kidney leukocytes after vaccination with Vibrio anguillarum. The IL-4/13 gene expression analysis after vaccination for 24h and 48h with 2 X 106 CFU ml-1 of inactivated Vibrio (Aquavac Vibrio Oral, ISPAH). mRNA levels were normalized to rRNA18 levels and calibrated against the non-stimulated 0 h control. Data were expressed as the mean ± SD. * indicates p < 0.05. The expression of IL-4/13 isoforms in sea bass vaccinated with NNV decreased from 24h to 48 h after the treatment, whereas in sea bass vaccinated with Vibrio anguillarum IL-4/13 isoforms transcript levels were highly up-regulated after 24h. In sea bass, we observed that the IL-4/13 isoforms in head kidney leukocytes seem to be modulated by the bacterial stimulus and, therefore, these cytokines might be useful markers of the immune responses against bacterial pathogens. Whang et al. (2016) have recently suggested that the expression of IL-4/13 isoforms in fish could be used as a novel molecular marker in evaluating vaccine efficacy in aquaculture. In conclusion, in this study we have addressed two issues related to the adaptive immune responses in sea bass. The obtained results are of interest both in relation the evolution of the immune system in vertebrates and for practical applications in aquaculture.I pesci Teleostei costituiscono un corposo gruppo con più di 20000 specie conosciute, in confronto alle 10000 specie di uccelli, e solo circa 5700 specie di mammiferi. I Teleostei sono adattati a differenti ambienti come, ad esempio, i Perciformi che sono presenti sia in acqua dolce sia in ambienti marini, tra cui l’Antartide. I pesci possono essere infettati da virus (rhabdoviruses, bornaviruses, reoviruses, nodaviruses, iridoviruses, herpesviruses, etc.), batteri (Vibrio, Aeromonas, Flavobacterium, Yersinia, Lactococcus, Mycobacterium, etc.) e molti altri parassiti. Dunque, ci si aspetta che una notevole diversità delle interazioni ospite/patogeno caratterizzano i meccanismi di difesa del sistema immunitario dei pesci. La spigola europea o branzino (Dicentrarchus labrax L.) è una specie marina di grande importanza economica, in particolar modo nell’acquacoltura del Mediterraneo. Tuttavia, numerosi virus, batteri, e altri patogeni attaccano questa specie, causando varie patologie che possono determinare massive perdite in acquacoltura. I linfociti B e T sono tra le principali cellule che mediano le risposte specifiche dell’immunità adattativa nei vertebrati con mascella. Una delle funzioni classiche delle cellule B è la produzione di anticorpi (o immunoglobuline) specialmente per neutralizzare i patogeni. Le immunoglobuline (Igs) sono fattori importanti del sistema immunitario adattativo e fino ad oggi, sono stati trovati in tutti gli Gnatostomi (Flajnik e Kasahara, 2010). Nei vertebrati sono stati individuati diversi isotipi di Igs mentre nei pesci Teleostei, fino a circa 10 anni fa, erano stati ritrovati, solo due isotipi di Igs: le IgM, una molecola tetramerica utilizzata principalmente per verificare la risposta immunitaria specifica contro i patogeni (Sunyer, 2012) e le IgD, una immunoglobulina monomerica la cui funzione ancora deve essere caratterizzata completamente (Edholm et al., 2011). Nel 2005 una nuova Ig è stata trovata, contemporaneamente, sia in trota iridea (Onchorynchus mykiss) che nel pesce zebra (Danio rerio) e denominata, rispettivamente, IgT (Hansen et al., 2005) e IgZ (Danilova et al., 2005). Successivamente, altre sequenze IgT/IgZ sono state identificate in diverse altre specie di pesci. La caratterizzazione funzionale di IgT è stata effettuata la prima volta nella trota arcobaleno (Onchorynchus mykiss) ed tale molecola è considerata principalmente coinvolta nella immunità mucosale (Zhang et al, 2010). La prima parte di questa tesi di Dottorato è stata focalizzata nello studio delle IgT in spigola. Nel trascrittoma di branchie di spigola, a disposizione nel nostro laboratorio (Nunez Ortiz et al., 2014), abbiamo identificato diverse sequenze parziali di IgT che ci hanno permesso di disegnare diversi primers utili per il clonaggio iniziale di questa molecola. Successivamente, mediante 3’ e 5’ RACEPCR, è stato possibile effettuare la ricostruzione dell’intera sequenza codificante la catena pesante dell’IgT secreta di spigola (accession number KM410929), che è risultata formata da 552 amminoacidi. Il dominio variabile (V) della IgT di spigola, presenta i tipici amminoacidi conservati in tutte le sequenze delle immunoglobuline e dei recettori delle cellule T (Lefranc 1999, Lefranc et al., 2003). Inoltre, l’intero dominio costante (C) risulta essere diviso in quattro domini CH come avviene nella maggior parte delle catene pesanti delle IgT. In spigola i quattro domini costanti (CH1-CH4) contengono i residui di cisteina e di triptofano necessari per il folding corretto dei domini immunoglobulinici (IgSF) (Lesk and Chothia, 1982). Il modello 3D dell’IgT di spigola è stato ottenuto mediante modellamento comparativo dopo aver selezionato il migliore modello disponibile in PDB. La struttura del modello preso in esame è identificata dal codice PDB: 1IGT, anticorpo monoclonale Ig gamma-2 A di topo. Spigole giovanili non stimolate sono state utilizzate per studiare i livelli dei trascritti delle IgT e delle IgM in differenti organi e tessuti. Elevati livelli di trascrizione delle IgT sono stati osservati nel muscolo, seguito da intestino, branchie e timo; mentre la massima espressione delle IgM è stata ritrovata nel cervello, seguito da milza, timo e fegato. Inoltre i livelli dei trascritti delle IgT nell’intestino e nelle branchie sono, in generale, molto più elevati rispetto ai livelli di espressione dei trascritti delle IgM (Figura 1). Ciò suggerisce che le IgM presentano un ruolo nelle risposte immunitarie sistemiche, mentre le IgT potrebbero essere specializzate nella immunità mucosale, come già dimostrato per la trota arcobaleno (Zhang et al., 2010). Figura 1 Espressione basale dei trascritti IgT ed IgM in tessuti differenti. I livelli di mRNA dei trascritti IgT ed IgM sono espressi come un rapporto riferito ai livelli di rRNA 18S nello stesso campione dopo analisi mediante PCR quantitativa, usando come calibratore il tessuto con il più basso livello di espressione (cervello) indicato dalla riga in nero. I dati sono espressi come la media ± SD da quattro differenti pesci. ** indicano che le IgT sono significativamente differenti dalle IgM con p< 0.01, mentre *** indicano che le IgT sono significativamente differenti dalle IgM con p