Bony Fish Gilthead Seabream Research Articles

Natural and synthetic estrogens modulate the inflammatory response in the gilthead seabream (Sparus aurata L.) through the activation of endothelial cells

Sex steroids are known to deeply alter processes other than fish reproduction, including fish growth, intermediary metabolism, osmoregulation and immunity. We have previously reported that 17β-estradiol (E2), the main fish estrogen, promotes the mobilization of acidophilic granulocytes from the head kidney, the bone marrow equivalent in fish, to the gonad in the bony fish gilthead seabream (Sparus aurata L.). The aim of this study was to investigate the effects of E2 and 17α-ethinylestradiol (EE2), an endocrine disruptor with strong estrogenic effects commonly found in the aquatic environment, on the ability of gilthead seabream endothelial cells (ECs) to promote leukocyte infiltration. E2 and EE2 were seen to affect ECs in different ways. Thus, E2 was able to increase the production of nitric oxide (NO) and up-regulate the expression of the key activation markers, interleukin-1β, CC chemokine ligand 4, interleukin-8, E-selectin and matrix metalloproteinase 9, when used alone or combined with bacterial DNA. In contrast, EE2 failed to affect NO release and reduced the up-regulation of the above genes promoted by bacterial DNA. Moreover, we found that leukocyte adhesion to ECs was enhanced by E2 treatment. Collectively, these results suggest that estrogens modulate fish leukocyte trafficking during an inflammatory process by activating ECs.

Estrogen-responsive genes in macrophages of the bony fish gilthead seabream: A transcriptomic approach

The role of sex steroids in the modulation of fish immune responses has received little attention. Previous studies have demonstrated that 17β-estradiol (E 2) is able to alter the response of gilthead seabream leukocytes to infectious agents. We have used suppression subtractive hybridization to identify genes upregulated by E 2 (50 ng/ml) in macrophage cultures from gilthead seabream. We isolated 393 up-regulated cDNA fragments that led to the identification of 162 candidate estrogen-responsive genes. Functional analyses revealed the presence of several enriched immune processes and molecular pathways. The E 2 up-regulation of some immune-relevant genes was further confirmed by real time RT-PCR. Bioinformatics analysis revealed the ability of E 2 to orchestrate profound alterations in the macrophage expression profile, especially immune-related processes and pathways. This is the first report on E 2-dependent modifications of fish macrophage transcriptome and lends weight to a suggested role for estrogen in the immune system, the possible significance of which is discussed.

TLR agonists extend the functional lifespan of professional phagocytic granulocytes in the bony fish gilthead seabream and direct precursor differentiation towards the production of granulocytes

Neutrophils are major cells participants in innate host responses. They are short-lived leukocytes, although microbial products activate intracellular signaling cascades that prolong their survival by inhibiting constitutive apoptosis. To gain insight into the phylogeny of this important cell type, we examined the ability of toll-like receptor agonists to extend the lifespan of gilthead seabream (Sparus aurata L.) acidophilic granulocytes, which are the functional equivalent of mammalian neutrophils. The results obtained demonstrated that apoptosis was also the default state of seabream acidophilic granulocytes and that toll-like receptor agonists were able to dramatically extend their functional lifespan (up to 10 days) by inhibiting apoptosis and inducing a long lasting activation of phagocytic and respiratory burst activities, together with the expression of genes coding for several proinflammatory molecules. This process was independent on contaminating cells and interleukin-1β production. In addition, the results showed that p38 mitogen-activated protein kinase, but not nuclear factor κB, c-Jun terminal kinase or phosphatidylinositol 3-kinase, was involved in the inhibition of acidophilic granulocyte apoptosis following toll-like receptor engagement. Finally, stimulation of head kidney hematopoietic precursor cells with toll-like receptor agonists promoted their terminal differentiation to acidophilic granulocytes. These results demonstrated that the extension of neutrophil lifespan by microbial products is conserved in lower vertebrates although the magnitude of the response is much higher in fish.

Prolactin-Induced Production of Reactive Oxygen Species and IL-1β in Leukocytes from the Bony Fish Gilthead Seabream Involves Jak/Stat and NF-κB Signaling Pathways

Prolactin (PRL), a peptide hormone produced by the pituitary gland, was shown to play an important role in the modulation of the immune system of lower and higher vertebrates. To further investigate the effects of PRL on the activation of professional phagocytes of bony fish, we stimulated head kidney leukocytes and purified macrophages from the gilthead seabream (Sparus aurata L.) with various physiological concentrations of native salmon PRL for 2 and 16 h and analyzed the respiratory burst activity and proinflammatory cytokine expression profile. The results showed that PRL was able to induce the production of reactive oxygen species and the expression of IL-1β and TNF-α in a similar way to two major pathogen-associated molecular patterns: polyinosinic-polycytidylic acid and genomic DNA from the bacterium Vibrio anguillarum. Interestingly, when the leukocytes were stimulated with suboptimal concentrations of PRL in the presence of bacterial DNA, the expression of IL-1β was synergistically induced. More importantly, all PRL activities were blocked by neutralizing Abs to PRL, as well as by pharmacological inhibitors of the Jak/Stat and NF-κB signaling pathways. In addition, EMSA and HPLC/mass spectrometry further confirmed that Stat and NF-κB were involved in the activation of seabream leukocytes by PRL. Collectively, our data identified PRL as a key regulator of the activation of fish professional phagocytes and demonstrated a cross-talk between TLR/NF-κB and PRLR/Jak/Stat signaling pathways. In addition, to the best of our knowledge, this is the first report showing that PRL modulates the activation of phagocyte NADPH oxidase through the Jak/Stat pathway in vertebrates.

Collagen regulates the activation of professional phagocytes of the teleost fish gilthead seabream

The innate immune system mediates the initial inflammatory response that follows infection or injury. Although the innate immune response of fish to infection has been relatively well characterized during recent years at both cellular and molecular levels, no studies have examined the role of extracellular matrix (ECM) in the regulation of innate immunity and inflammation. We report here that collagen and gelatin in vitro were able to prime the respiratory burst of phagocytes from the bony fish gilthead seabream. In addition, collagen and gelatin induced a specific set of immune-related and ECM remodelling enzymes that substantially differed from that induced by pathogen-associated molecular patterns. Notably, both collagen and gelatin induced the expression of interleukin-1β, chemokine (C–C motif) ligand 4 and matrix metalopeptidases (MMP) 9 and 13 in acidophilic granulocytes and macrophages but were unable to significantly increase the expression of other pro-inflammatory genes. Furthermore, it was found that the MMP2/MMP9 inhibitor V had a dose-dependent inhibitory effect on seabream phagocyte activation by either collagen or gelatin. In contrast, pre-treatment of collagen and gelatin by collagenase resulted in a higher stimulatory capacity compared to non-digested proteins. Collectively, these results indicate that collagen fragments produced by the action of different host proteases, and probably released by infectious agents, are sensed by fish phagocytes. Therefore, we propose that, besides to the well-established response to infection, the innate immune system of fish is able to respond to tissue injury.

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 α and β 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.

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.

The antimicrobial peptide hepcidin exerts an important role in the innate immunity against bacteria in the bony fish gilthead seabream

Antimicrobial peptides (AMPs) are important mediators of the immune response against bacteria and hepcidin is a 20–25 residues member with known functions in iron regulation and the innate immune response. Most studies have focused on mammalian organisms but very little is known about other vertebrate groups including teleost fish. Thus, based on the sequence of an EST database, we have characterized hepcidin gene organization, gene expression, distribution and in vitro and in vivo regulation, as well as the biological activity of a synthetic peptide in the teleost fish gilthead seabream ( Sparus aurata L.). First, it was found that the seabream hep gene genomic organization is formed by 3 exons and 2 introns, while the mRNA transcript is constitutively detected in most of the fish tissues but mainly in peritoneal leucocytes, head-kidney, liver and skin. Moreover, we have identified for the first time that hep is much more highly expressed in acidophilic granulocytes than in monocyte-macrophages and lymphocytes. In vitro, hep expression is up-regulated by several mitogens, pathogen-associated molecular patterns (PAMPs) and particulated antigens. Not surprisingly, intraperitoneal injection of bacteria or virus led to a significant gene up-regulation in the liver, head-kidney, peritoneal exudate or spleen. These observations suggest a major role for seabream hepcidin in the immune response to bacteria and viruses. Furthermore, the synthetic seabream Hep exerted an important antimicrobial activity against several bacterial strains in vitro reducing their viability. To conclude, seabream hep gene expression, up-regulation after in vitro or in vivo treatment with mitogens, PAMPs or particulated antigens and the direct in vitro biological activity against bacteria demonstrate that it is an important antimicrobial peptide and probably plays an important role in the innate immune response of fish.

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-1β (IL-1β), 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.

Mechanisms of processing and secretion of IL-1β in fish (94.30)

Abstract IL-1β is a potent pro-inflammatory cytokine which is translated as a leaderless 33 kDa precursor protein (proIL-1β) that is processed into its 17 kDa active form by the IL-1β-converting enzyme (ICE, caspase-1). Several stimuli have been reported to activate caspase-1, including the engagement of P2X7 receptors by extracellular ATP and infection with invasive bacteria. While the recognition site for caspase-1 is well conserved in mammals, most of the non-mammalian IL-1β genes cloned so far lack this conserved site. In addition, we have previously shown that extracellular ATP fails to induce the processing and release of this cytokine in the bony fish gilthead seabream. In this study, we report that the stimulation of seabream leukocytes with flagellin or bacterial DNA leads to the processing of proIL-1β into a 20 kDa form, although it largely remains intracellular. However, the infection of leukocytes with salmonella results in the efficient and specific release of the mature form. Interestingly, the processing of IL-1β in this species is not prevented by a specific inhibitor of caspase-1 or a pan-caspase inhibitor. Whatever the case, our results suggest that the processing and release of IL-1β in early vertebrates is mediated by cell death upon bacterial infection by a caspase-1 independent mechanism, while a more sophisticated mechanism operated by activation of ATP-gated P2X7 receptors appeared later in evolution. This work was supported by the Spanish Ministry of Education and Science (grant BIO2005-05078 to V.M. and fellowship to G.L.-C.).

Fish lymphocytes regulate the inflammatory response via the production of chemokines (94.24)

Abstract Macrophages provide the first line of defense against infections through the recognition of different pathogen-associated molecular patterns (PAMPs). Upon activation, they produce pro-inflammatory cytokines, such as interleukin-1β (IL-1β) and tumor necrosis factor α (TNFα), which regulate not only innate but also adaptive immunity. In the bony fish gilthead seabream, we have shown that TNFα produced by activated macrophages induces the recruitment and activation of professional phagocytic granulocytes when injected intraperitoneally, but it hardly activates these cells in vitro. In this study, we report that seabream TNFα is able to up-regulate the expression of E-selectin in endothelial cells, leading to a concomitant increased adhesion of activated granulocytes and lymphocytes. More importantly, although all fish immune cell types analyzed, i.e. granulocytes, macrophages and lymphocytes, produce the CCL4-like chemokine in response to different PAMPs, only lymphocytes and endothelial cells are able to produce this chemokine in response to TNFα. These results demonstrate that TNFα in fish is mainly involved in the recruitment of leukocytes to the inflammatory foci rather than in their activation and suggest a role for fish lymphocytes in the regulation of early immune responses to infection. This work was supported by the Spanish Ministry of Education and Science (grant BIO2005-05078) and the Sixth Framework Programme of the European Union (grant SSP8-CT-2003-501984).

The type II interleukin-1 receptor (IL-1RII) of the bony fish gilthead seabream Sparus aurata is strongly induced after infection and tightly regulated at transcriptional and post-transcriptional levels

Interleukin-1β (IL-1β) is the prototypic pro-inflammatory cytokine. All the biological effects of IL-1β are mediated through interaction with type 1 IL-1 receptor (IL-1RI), whereas another receptor, called type 2 IL-1R (IL-1RII), lacks an intracellular signalling domain and acts as a decoy receptor that down-regulates responses to IL-1β. Although both receptors are present in bony fish, their expression and biological role in the regulation of IL-1β activity in non-mammalian vertebrates remain to be established. In this study, a homologue of mammalian IL-1RII was isolated and characterized in the gilthead seabream (Sparus aurata). The seabream IL-1RII harboured two Ig-like domains in its extracellular region and a short cytoplasmic tail lacking a signalling domain. The seabream IL-1RII cDNA showed an unexpectedly long 3′UTR compared with that from other species and contained three ATTTA instability motifs, which seem to be responsible for its relatively short half-life (less than 2h). The expression of seabream IL-1RII was dramatically up-regulated after infection with Vibrio anguillarum in all the immune tissues examined and was even more strongly induced than the IL-1β gene in the head kidney, spleen and liver. Strikingly, the mRNA levels of IL-1RII were 15-fold higher than those of IL-1β in the liver, suggesting a role for this organ in the neutralization of IL-1β leaking into the systemic circulation from the sites of inflammation. In vitro, bacterial DNA and flagellin increased the mRNA levels of IL-1RII in macrophages, while only flagellin was able to weakly induce its expression in acidophilic granulocytes. Finally, the seabream IL-1RII was localized in the plasma membrane when expressed in HEK293 cells and was able to bind IL-1β.

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.

The colony-stimulating factor-1 receptor is a specific marker of macrophages from the bony fish gilthead seabream

We report the molecular cloning of the colony-stimulating factor-1 receptor gene from the bony fish gilthead seabream ( sbCSF-1R). The deduced sbCSF-1R shows a predicted signal sequence, a transmembrane domain and a tyrosine kinase domain, all in conserved positions. A transcript showing a premature stop codon that predicted the removal of 84 C-terminal amino acids was also found. RT-PCR expression studies demonstrate that, although the sbCSF-1R transcripts are found in different immune tissues, including gill, liver, spleen, blood, peritoneal exudate, thymus and head–kidney (HK), their expression is confined to the monocyte/macrophage lineage. Furthermore, the expression of sbCSF-1R might be modulated by the activation stage of the macrophages, since both the infection of fish and the in vitro activation of leukocytes resulted in the down-regulation of gene expression. These data indicate that the CSF-1R may be used as a specific probe for cells of the monocyte/macrophage lineage in the gilthead seabream, an immunological tractable fish model. In addition, the functional characterisation of the CSF-1R and its ligand may shed light into the mechanisms of proliferation and the pathways of differentiation of macrophages in bony fish.

Early innate immune response and redistribution of inflammatory cells in the bony fish gilthead seabream experimentally infected with Vibrio anguillarum

An obvious difference between the immune system of fish and mammals is that fish lack both bone marrow and lymph nodes; in their place, the head-kidney acts as a major haematopoietic and lymphoid organ in adult fish, whereas the thymus, spleen and mucosa-associated lymphoid tissues are common to both fish and mammals. This suggests that differences exist in antigen presentation and naive lymphocyte stimulation, a prerequisite for the initiation of adaptive immune responses. Intraperitoneal injection of the bony fish gilthead seabream (Sparus aurata L.) with intact Vibrio anguillarum, as a particulate bacterial antigen, results in the mobilisation of head-kidney leucocytes to the peritoneal cavity and priming of their respiratory burst activity. We have also observed the rapid infiltration of acidophilic granulocytes, which are leucocytes functionally equivalent to mammalian neutrophils, into the spleen. These cells may be involved in antigen capture and transport to the spleen, since an apparent association between mobilised acidophilic granulocytes, bacterial antigens and proliferating lymphocytes has been seen in this organ. Collectively, these results suggest that, in addition to being actively involved in bacterial clearance, fish phagocytic granulocytes play a role in the initiation and support of the adaptive immune response.

The tumor necrosis factor alpha of the bony fish seabream exhibits the in vivo proinflammatory and proliferative activities of its mammalian counterparts, yet it functions in a species-specific manner.

Information on the bioactivities of non-mammalian cytokines is scant due to the lack of the recombinant molecules and specific antibodies. We produced the mature predicted peptide of tumor necrosis factor alpha (TNF alpha) from the bony fish gilthead seabream (Sparus aurata L.) (sbTNF alpha), and its biological role was determined in vitro and in vivo. We first demonstrated by analytical size-exclusion chromatography that sbTNF alpha is an oligomeric protein but the dimer appears to predominate over the trimeric form, in contrast to mammalian TNF alpha. Intraperitoneal injection of native sbTNF alpha resulted in (i) priming of the respiratory burst of the peritoneal exudate and head-kidney (HK) leukocytes, the latter being the bone marrow equivalent in fish; (ii) rapid recruitment of phagocytic granulocytes to the injection site, and (iii) induction of granulopoiesis in the HK. Interestingly, sbTNF alpha was able to induce a strong proliferation of HK cells in vitro, whereas human TNF alpha did not. Conversely, sbTNF alpha was not cytotoxic for murine L929 fibroblasts.