Modulation of leukocytic populations of gilthead sea bream (Sparus aurata) by the intestinal parasite Enteromyxum leei (Myxozoa: Myxosporea).

SUMMARY The cellular mucosal and systemic effectors of gilthead sea bream (GSB) (Sparus aurata) involved in the acute immune response to the intestinal parasite Enteromyxum leei were studied in fish experimentally infected by the anal route. In the intestinal inflammatory infiltrates and in lymphohaematopoietic organs (head kidney and spleen) of parasitized fish, the number of plasma cells, B cells (IgM immunoreactive) and mast cells (histamine immunoreactive) were significantly higher, whereas the number of acidophilic granulocytes (G7 immunoreactive) decreased, compared with non-parasitized and unexposed fish. These differences were stronger at the posterior intestine, the main target of the parasite, and no differences were found in the thymus. In non-parasitized GSB, the percentage of splenic surface occupied by melanomacrophage centres was significantly higher. These results suggest that the cellular response of GSB to E. leei includes proliferation of leukocytes in lymphohaematopoietic organs and recruitment into intestines via blood circulation involving elements of innate and adaptive immunity. Acidophilic granulocytes and mast cells presented opposite patterns of response to the parasite infection, with an overall depletion of the former and an increased amount of the latter. Some differences between both cell types were also detected in regard to their granule density and cell morphology.

Recombinant TNFα as oral vaccine adjuvant protects European sea bass against vibriosis: insights into the role of the CCL25/CCR9 axis.

Vibrio anguillarum is the main causative agent of vibriosis in cultured sea bass. Unfortunately, available vaccines against this disease do not achieve the desired protection. In this study, to accomplish uptake, processing, and presentation of luminal antigens, a commercial sea bass oral vaccine against V. anguillarum was improved with the addition of recombinant fish-self tumor necrosis factor α (rTNFα), as adjuvant. To explore mechanisms, systemic and local responses were analyzed through serum specific IgM titers, gene expression, lymphocytes spatial distribution in the gut, and in vitro functional assays. We found along the trial, over expressed transcripts of genes encoding cytokines and antimicrobial molecules at the gut of rTNFα supplied group. Orally immunized fish with vaccine alone confer protection against V. anguillarum challenge throughout a short time period. In contrast, adjuvant-treated group significantly extended the response. In both cases, achieved protection was independent of serum IgM. Yet, IgT transcripts were found to increase in the gut of rTNFα-treated fish. More importantly, fish treated with rTNFα showed a dramatic change of their T lymphocytes distribution and localization in gut mucosal tissue, suggesting specific antigen recognition and further intraepithelial T lymphocytes (IEL) activation. To determine the mechanism behind IEL infiltration, we characterized the constitutive and activated pattern of chemokines in sea bass hematopoietic tissues, identifying for the first time in fish gut, an intimate relation between the chemokine ligand/receptor CCL25/CCR9. Ex-vivo, chemotaxis analyses confirmed these findings. Together, our results demonstrate that improved oral vaccines targeting key cytokines may provide a means to selectively modulate fish immune defence.

Evolution of the inflammatory response in vertebrates: fish TNF-alpha is a powerful activator of endothelial cells but hardly activates phagocytes.

TNF-alpha is conserved in all vertebrate classes and has been identified in all taxonomic groups of teleost fish. However, its biological activities and its role in infection are largely unknown. Using two complementary fish models, gilthead seabream and zebrafish, we report here that the main proinflammatory effects of fish TNF-alpha are mediated through the activation of endothelial cells. Thus, TNF-alpha promotes the expression of E-selectin and different CC and CXC chemokines in endothelial cells, thus explaining the recruitment and activation of phagocytes observed in vivo in both species. We also found that TLR ligands, and to some extent TNF-alpha, were able to increase the expression of MHC class II and CD83 in endothelial cells, which might suggest a role for fish endothelial cells and TNF-alpha in Ag presentation. Lastly, we found that TNF-alpha increases the susceptibility of the zebrafish to viral (spring viremia of carp virus) and bacterial (Streptococcus iniae) infections. Although the powerful actions of fish TNF-alpha on endothelial cells suggest that it might facilitate pathogen dissemination, it was found that TNF-alpha increased antiviral genes and, more importantly, had little effect on the viral load in early infection. In addition, the stimulation of ZF4 cells with TNF-alpha resulted in increased viral replication. Together, these results indicate that fish TNF-alpha displays different sorts of bioactivity to their mammalian counterparts and point to the complexity of the evolution that has taken place in the regulation of innate immunity by cytokines.

Histamine is stored in mast cells of most evolutionarily advanced fish and regulates the fish inflammatory response.

Mast cells are important as initiators and effectors of innate immunity and regulate the adaptive immune responses. They have been described in all classes of vertebrates and seem to be morphologically and functionally similar. However, early studies had shown that fish and amphibian mast cells were devoid of histamine. In this study, we take a fresh look at the evolution of histamine and find that the mast cells of fish belonging to the Perciformes order, the largest and most evolutionarily advanced order of teleosts, are armed with histamine. More importantly, histamine is biologically active in these fish where it is able to regulate the inflammatory response by acting on professional phagocytes. In addition, the actions of histamine in these immune cells seem to be mediated through the engagement of H(1) and H(2) receptors, which, together with the H(3) receptor, are well conserved in bony fish. We propose that the storage of histamine in vertebrate mast cells and its use as an inflammatory messenger was established in primitive reptiles (Lepidosauria) approximately 276 million years ago. This same feature seems to have developed independently in Perciform fish much more recently in the Lower Eocene, between 55 and 45 million years ago, a short period during which the great majority of Percomorph families appeared.

Molecular and functional characterization of gilthead seabream Sparus aurata caspase-1: the first identification of an inflammatory caspase in fish.

Caspases are a family of cysteine proteases that fulfil critical roles in mammalian apoptosis and in the proteolytic activation of cytokines. In humans, the caspase family includes 13 members whose functions seem to correlate with their phylogenetic relationship. They are classified into two main groups, the cell death (apoptotic) and the inflammatory caspases. Caspase-1 is the best characterized inflammatory caspase and is responsible for the processing of interleukin-1beta (IL-1beta), IL-18 and IL-33. Despite the importance of caspase-1 in inflammation, no information is available on the presence and activity of this enzyme in fish. In this study, we cloned a caspase-1-like gene from the bony fish gilthead seabream (Sparus aurata L.) which shows a conserved N-terminal caspase-recruitment domain (CARD) and a C-terminal caspase catalytic domain. The seabream caspase-1 gene was expressed in 1 day post-hatching larvae and its mRNA levels increased throughout development. In adult fish, caspase-1 was found to be constitutively expressed in all immune tissues analyzed and, unexpectedly, infection of fish and stimulation of professional phagocytes in vitro decreased its mRNA levels. It was also demonstrated that the recombinant seabream caspase-1 ectopically expressed in HEK293 cells was able to cleave a caspase-1 specific substrate, this activity being enhanced upon activation of the rat P2X7 receptor with BzATP. Finally, seabream fibroblast cell line SAF-1 and primary leukocytes showed endogenous caspase-1 activity, which was almost completely inhibited by a caspase-1 specific inhibitor.

Vaccination of larvae of the bony fish gilthead seabream reveals a lack of correlation between lymphocyte development and adaptive immunocompetence.

Fish eggs are released and embryos hatch into a pathogenically hostile environment, at a time when their immunological capacity is severely limited. Although the eggs are initially protected by the envelope as well as by several innate and adaptive immune substances, which are transferred to eggs during fish vitellogenesis, it seems that young specimens depend fundamentally on their innate defence mechanisms. Here we show in the gilthead seabream, an immunologically tractable teleost fish model, that the first lymphocyte marker genes, those coding for the two subunits for the recombination activating gene, were detected by RT-PCR around 21-27 days post-hatching (dph). In addition, the transcripts coding for the alpha and beta subunits of the T-cell receptor and the light and heavy chains of immunoglobulin M were detected at 27-48 dph. However, most innate immune genes analyzed were already expressed at hatching, including those coding for the toll-like receptors, pro- and anti-inflammatory molecules, antiviral and antibacterial factors, and phagocyte markers. Using the information from the gene expression study, we also examined the achievement of immunocompetence by analyzing the protection induced by a bacterin against the pathogenic bacterium Photobacterium damselae subsp. piscicida. The results show that vaccination of young larvae of this species by either immersion or oral routes resulted in increased susceptibility to infection of the specimens, and point to the lack of correlation between the achievement of immunocompetence and detection of the adaptive immunity markers.

The antimicrobial peptides piscidins are stored in the granules of professional phagocytic granulocytes of fish and are delivered to the bacteria-containing phagosome upon phagocytosis.

Antimicrobial peptides (AMPs) are increasingly recognized as a critical first line of defence against many pathogens. The genes encoding these peptides are expressed in numerous tissue and cell types from a wide variety of different species including mammals, amphibians, fish, and insects. In this study, we report that the AMPs called piscidins were primarily present in the mast cells (MCs) of fish and were only identified in fish belonging to the Order Perciformes. It is striking that histamine was seen to have a similar evolutionary history, since the only piscine MCs endowed with this molecule are in the Perciformes. We also show that both MCs and professional phagocytic granulocytes were armed with different piscidin molecules. In contrast, macrophages were devoid of these AMPs. More importantly, we found by immunoelectron microscopy that piscidins were delivered to the bacteria-containing phagosome of granulocytes upon phagocytosis, suggesting a role for these AMPs in the killing of both extracellular and intracellular pathogenic bacteria.

Characterization of macrophages from the bony fish gilthead seabream using an antibody against the macrophage colony-stimulating factor receptor.

Two major professional phagocyte populations have been described in fish, namely granulocytes and monocytes/macrophages. Although the distribution and localization of macrophages have been documented in several teleost species using mainly light and/or electron microscopy, the lack of appropriate markers for these cells has hampered our in-depth knowledge of their biology. We report here the generation of a monospecific rabbit polyclonal antibody against the gilthead seabream macrophage colony-stimulating factor receptor (Mcsfr), which is an excellent marker of macrophages in mammals and the zebrafish. The anti-Mcsfr has been found to be very useful in immunohistochemistry (IHC) to specifically immunostain the purified macrophages (adherent cells) obtained from the head-kidney as well as different cell populations in paraffin-embedded organs, including the head-kidney, spleen, thymus, gills and liver. Unexpectedly, however, no Mcsfr immunoreactive (Mcsfr(+)) cells were observed in the brain and intestine of the gilthead seabream. We also show that the distribution of Mcsfr(+) cells in the head-kidney and the spleen is unaltered following infection with the fish pathogenic bacterium Vibrio anguillarum and that the Il1b-producing cells in these two organs after infection are exclusively acidophilic granulocytes. Finally, as the epitope recognized by the anti-Mcsfr is well conserved, we illustrate the potential usefulness of this antibody in other teleost species, such as the European seabass.

Distribution of the professional phagocytic granulocytes of the bony fish gilthead seabream (Sparus aurata L.) during the ontogeny of lymphomyeloid organs and pathogen entry sites.

Although it is believed that fish fry depend fundamentally on their innate defence mechanisms, the ontogeny of fish innate immune cells is poorly understood. In the present study, we have used a specific monoclonal antibody against acidophilic granulocytes (AGs), the main professional phagocytic cell type of the bony fish gilthead seabream, to study their localization during the development of the main lymphomyeloid organs, namely the head kidney, spleen and thymus, and of the two major portals for pathogen entry, namely the gills and intestine. AGs were observed in the posterior intestine and in the blood earlier than in the haematopoietic kidney (21 vs. 27 days post-hatching, dph). AGs were observed scattered between other cells of the haematopoietic lineage in the head kidney of larvae, but were grouped around the blood vessels of this organ in juveniles and adults, where they were also much more numerous. In the spleen and in the thymus, AGs were observed much later (62 dph) and appeared scattered. AGs were also observed in the gill lamella and the posterior intestine near the anus throughout development.

Detection of antimicrobial peptides related to piscidin 4 in important aquacultured fish.

Epithelial surfaces of fish, such as the gut, skin and gills, comprise a large surface area for possible pathogen invasion. Antimicrobial peptides (AMPs), innate immunity components, play a significant role in protecting fish. Piscidins are a family of AMPs. In this study, we detected the presence of the recently discovered piscidin 4 via bug blot, Western blot, ELISA and/or immunohistochemistry in striped bass (Morone saxatilis), white bass (M. chrysops), European seabass (Dicentrarchus labrax), gilthead seabream (Sparus aurata), red drum (Sciaenops ocellatus), and barramundi (Lates calcarifer). Via bug blot, gill extracts from all species had antibacterial activity corresponding to the migration rate of piscidin 4. Western blotting showed that piscidin 4 immunoreactivity was greatest in striped bass gill extract. The concentrations of piscidin 4 detected by the ELISA in striped bass gill (approximately 20 microg/ml) were well within the levels that are inhibitory to important fish bacterial pathogens. Piscidin 4 was also detected via immunohistochemistry in all fish except barramundi. Piscidin 4-positive cells were identified as mast cells (MC), but not all MC were piscidin 4-positive. Species, age, size and physiological condition at sampling were some factors that might affect piscidin expression in different species. Our data provide strong evidence that piscidin 4 isoforms are present in all these commercially important species.

Early presence of immune cells in the developing gonad of the gilthead seabream (Sparus aurata Linnaeus, 1758).

A great deal is known regarding the process of sex differentiation in fish. However, little is known about the presence of immune cells and cytokines in this process. In the gilthead seabream, both immune cells and cytokines play an important role in the tissue reorganization of the gonads during the adult reproductive cycle. We have studied, using light microscopy and immunocytochemistry, the ontogenetic development of the gilthead seabream gonads, focusing on the presence of immune cells and cytokines. We show that the testicular area is quickly differentiated and becomes functional in specimens less than a year old, while the ovarian area differentiates later and continues to develop during the first two years of life. Throughout the morphogenesis process, acidophilic granulocytes were present in the gonad. Interleukin-1b (Il1b) is produced in the testicular area in juveniles and male fish, but not in the ovarian area. Macrophage-colony stimulating factor receptor (Mcsfr) is not produced in the undifferentiated gonad and is only found once the testicular area is well developed.