Extending immunological profiling in the gilthead sea bream, sparus aurata, by enriched cDNA library analysis, microarray design and initial studies upon the inflammatory response to PAMPs

This study describes the development and validation of an enriched oligonucleotide-microarray platform for Sparus aurata (SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene expression in S. aurata with an emphasis upon immunity and the immune response

Cimetidine disrupts the renewal of testicular cells and the steroidogenesis in a hermaphrodite fish

The importance of histamine in the physiology of the testis in mammals and reptiles has been recently shown. Histamine receptors (Hrs) are well conserved in fish and are functional in several fish species. We report here for the first time that histamine and the mRNA of Hrh1, Hrh2 and Hrh3 are all present in the gonad of the hermaphrodite teleost fish gilthead seabream. Moreover, cimetidine, which acts in vitro as an agonist of Hrh1 and Hrh2 on this species, was intraperitoneally injected in one and two years old gilthead seabream males. After three and five days of cimetidine injection, we found that this compound differently modified the gonadal hrs transcript levels and affects the testicular cell renewal and the gene expression of steroidogenesis-related molecules as well as the serum steroid levels. Our data point to cimetidine as a reproductive disruptor and elucidate a role for histamine in the gonad of this hermaphrodite fish species through Hr signalling.Postprint2,616

Flow cytometry based techniques to study testicular acidophilic granulocytes from the protandrous fish gilthead seabream (Sparus aurata L.)

The gilthead seabream is a protandrous seasonal breeding teleost that is an excellent model for studying the testicular regression process which occurs in both seasonal testicular involution and sex reversion. Little is known about the cell types and the molecular mechanisms involved in such processes, mainly because of the lack of appropriate methods for testis dissociation, and testicular cell isolation, culture and functional characterization. We have previously reported that gilthead seabream acidophilic granulocytes infiltrate the testis at post-spawning stage, settle close to the spermatogonia and accumulate intracellular interleukin-1β. In this paper, we report several flow cytometry based assays which allow to establish the role played by gilthead seabream testicular acidophilic granulocytes and permits their quantification

Aluminum as vaccine adjuvant increases immunity, but deplete splenic melanomacrophage centers in sea bream

Aluminum as vaccine adjuvant has been proved to be successful in several species at inducing increased immune responses against the many poorly immunogenic antigens. Fish are not the exception and promising results with different antigens in many species are on-demand. Even though, the mechanism of action of this adjuvant often remains unclear. In this study, we tested by IP a commercial formulation of aluminum hydroxide and magnesium (Imject Alum) as adjuvant of the model antigen keyhole limpet hemocyanin (KLH), and found that it has as a potent and unique adjuvancy capacity. Compared to control group, two commercial preparations (Montanide 761 or 763) or KLH alone, Imject Alum plus KLH formula, despite the powerful immunogenic activity recorded over the innate (IL-1b expression and serum lysozyme)and adaptive (total serum and skin mucus IgM) immune responses throughout the 8 months of trial, it also caused several damages at the injection site. Among damages, severe adhesions, granulomatosis and a transitory melanization were recorded, but, without obvious signs of impaired animal health. Additionally, optical screening for external tissue damage was performed, but any change was observed. Surprisingly, histopathology evaluation trough classical stains of head-kidney, spleen, liver and gut revealed that melanomacrophage centers at the spleen were depleted of cells, but not in HK. Furthermore TEM confirmed this data which suggest iron retention in MMCs or a high catabolic activity exerted by erythrocytes as possible mechanisms associated with depletion. Whatever the case, the use of aluminum as vaccine adjuvant must be further explored in detail before the commercial application in aquaculture facilities

Role of estrogens in fish immunity with special emphasis on GPER1

It is well accepted that estrogens, the primary female sex hormones, play a key role in modulating different aspects of the immune response. Moreover, estrogens have been linked with the sexual dimorphism observed in some immune disorders, such as chronic inflammatory and autoimmune diseases. Nevertheless, their effects are often controversial and depend on several factors, such as the pool of estrogen receptors (ERs) involved in the response. Their classical mode of action is through nuclear ERs, which act as transcription factors, promoting the regulation of target genes. However, it has long been noted that some of the estrogen-mediated effects cannot be explained by these classical receptors, since they are rapid and mediated by non-genomic signaling pathways. Hence, the interest in membrane ERs, especially in G protein-coupled estrogen receptor 1 (GPER1), has grown in recent years. Although the presence of nuclear ERs, and ER signaling, in immune cells in mammals and fish has been well documented, information on membrane ERs is much scarcer. In this context, the present manuscript aims to review our knowledge concerning the effect of estrogens on fish immunity, with special emphasis on GPER1. For example, the numerous tools developed over recent years allowed us to report for the first time that the regulation of fish granulocyte functions by estrogens through GPER1 predates the split of fish and tetrapods more than 450 million years ago, pointing to the relevance of estrogens as modulators of the immune responses, and the pivotal role of GPER1 in immunity

Professional phagocytic granulocyte-derived PGD2 regulates the resolution of inflammation in fish

Prostaglandins (PGs) play a key role in the development on the immune response through the regulation of both pro- and anti-inflammatory processes. PGD2 can be either pro- or anti-inflammatory depending on the inflammatory milieu. Prostaglandin D synthase (PGDS) is the enzyme responsible for the conversion of PGH2 to PGD2. In mammals, two types of PGDS synthase have been described, the hematopoietic (H-PGDS) and the lipocalin (L-PGDS). In the present study we describe the existence of two orthologs of the mammalian L-PGDS (PGDS1 and PGDS2) in the gilthead seabream and characterize their gene expression profiles and biological activity. The results showed a dramatic induction of the gene coding for PGDS1 in acidophilic granulocytes (AGs), which are functionally equivalent to mammalian neutrophils, after a prolonged in vitro activation with different pathogen associated molecular patterns (PAMPs). In contrast PGDS2 was not expressed in these cells. The functional relevance of the induction of PGDS1 in AGs was confirmed by the ability of these cells to release PGD2 upon PAMP stimulation. To gain further insight into the role of PGD2 in the resolution of inflammation in fish, we examined the ability of PGD2 or its cyclopentenone derivates (cyPGs) to modulate the main functional activities of AGs. It was found that both PGD2 and cyPGs inhibited the production of reactive oxygen species and downregulated the transcript levels of the gene encoding interleukin-1β. Taken together, these results demonstrate that the use of PGD2 and its metabolites in the resolution of inflammation was established before the divergence of fish from tetrapods more than 450 million years ago and support a critical role for granulocytes in the resolution of inflammation in vertebrates.Versión del editor3,268

Transcriptomic analysis of immune-relevant genes in the gilthead seabream (Sparus aurata)

4 pages, 2 figures, 1 table.-- XI Congreso Nacional de Acuicultura, Vigo 25-28 de septiembre de 2007[EN] To date, immune-relevant nucleotide and protein sequences for the seabream (Sparus aurata) are not well represented in currently available sequence databases. The most likely reason is because expressed sequence tag (EST) projects that set out to identify immune-related genes are developed by sequencing cDNA libraries from non-stimulated immune cells or tissues. Regulation of immune factors is under a strict control and their expression is only regulated when immune cells are stimulated by PAMPs (Pathogen Associated Molecular Patterns). In order to increase the number of immune-relevant sequences in seabream, a cDNA library was constructed with several immune cells and tissues stimulated with different PAMPs[ES] Hasta el momento, tanto las secuencias nucleotídicas y proteicas inmuno relevantes para la dorada (Sparus aurata) no están bien representadas en las bases de datos existentes. La razón es porque los proyectos de EST (Expressed Sequence Tag) que intentan identificar genes inmunes relacionados son realizados a partir de la secuenciación de librerías de cDNA de células inmunes y tejidos no estimulados. La regulación de los factores inmunes está bajo un control estricto y su expresión sólo es regulada cuando las células inmunes son estimuladas con PAMPs (Patrones Moleculares Asociados a Patógenos). Para poder obtener un incremento en el número de secuencias inmuno relevantes en dorada se construyó una librería de cDNA a partir de múltiples células y tejidos inmunes estimulados con diferentes PAMPsN

Isolation of mast cells from the peritoneal exudate of the teleost fish gilthead sea bream (Sparus aurata L.)

Inflammation is the first response of animals to infection or tissue damage. Sparus aurata (Perciformes) was the first fish species shown to possess histamine-containing mast cells at mucosal tissues. We report a separation protocol for obtaining highly enriched (over 95% purity) preparations of fish mast cells in high numbers (5-20 million mast cells per fish). The peritoneal exudate of S. aurata is composed of lymphocytes, acidophilic granulocytes, macrophages and mast cells. We separated the lymphocyte fraction through discontinuous density gradient centrifugation. The remaining cells were cultivated overnight in RPMI-1640 culture medium containing 5% fetal calf serum, which allowed macrophages to adhere to the cell culture flasks. Finally, acidophilic granulocytes were separated from the mast cells though a Magnetic-Activated Cell Separation (MACS) protocol, using a monoclonal antibody against these cells. The purity of mast cells-enriched fractions was analyzed by flow cytometry and by transmission electron microscopy. The functionality of purified mast cells was confirmed by the detection of histamine release by ELISA after stimulation with compound 48/80 and the induction of the pro-inflammatory cytokines IL-1β and IL-8 following stimulation with bacterial DNA. This fish mast cells separation protocol is a stepping stone for further studies addressing the evolution of vertebrate inflammatory mechanisms.Postprin