Joan Carles Balasch

RNA-Seq Reveals an Integrated Immune Response in Nucleated Erythrocytes

Background Throughout the primary literature and within textbooks, the erythrocyte has been tacitly accepted to have maintained a unique physiological role; namely gas transport and exchange. In non-mammalian vertebrates, nucleated erythrocytes are present in circulation throughout the life cycle and a fragmented series of observations in mammals support a potential role in non-respiratory biological processes. We hypothesised that nucleated erythrocytes could actively participate via ligand-induced transcriptional re-programming in the immune response. Methodology/Principal Findings Nucleated erythrocytes from both fish and birds express and regulate specific pattern recognition receptor (PRR) mRNAs and, thus, are capable of specific pathogen associated molecular pattern (PAMP) detection that is central to the innate immune response. In vitro challenge with diverse PAMPs led to de novo specific mRNA synthesis of both receptors and response factors including interferon-alpha (IFNα) that exhibit a stimulus-specific polysomal shift supporting active translation. RNA-Seq analysis of the PAMP (Poly (I∶C), polyinosinic∶polycytidylic acid)-erythrocyte response uncovered diverse cohorts of differentially expressed mRNA transcripts related to multiple physiological systems including the endocrine, reproductive and immune. Moreover, erythrocyte-derived conditioned mediums induced a type-1 interferon response in macrophages thus supporting an integrative role for the erythrocytes in the immune response. Conclusions/Significance We demonstrate that nucleated erythrocytes in non-mammalian vertebrates spanning significant phylogenetic distance participate in the immune response. RNA-Seq studies highlight a mRNA repertoire that suggests a previously unrecognized integrative role for the erythrocytes in other physiological systems.

The effects of immunostimulation through dietary manipulation in the rainbow trout; evaluation of mucosal immunity.

Immunostimulant-containing diets are commonly used in aquaculture to enhance the resistance of cultured fish to disease and stress. Although widespread in use, there have been conflicting results published, and surprisingly little is known about the regulation of immune response-related genes in tissues key to mucosal immunity induced by immunostimulant dietary feeding. Using a salmonid-specific microarray platform enriched with immune-related genes and in situ hybridization, we investigated dietary acclimation in two organs relevant to mucosal immunity, the gills and the intestine, in the rainbow trout (Oncorhynchus mykiss). Immunostimulant diets significantly changed gene expression profiles and gene distribution in a tissue-specific manner: genes and functional Gene Ontology categories involved in immunity were differently expressed at portals of entry where significant changes in genes and functional groups related to remodeling processes and antigen presentation were observed. Furthermore, genes involved in chemotaxis, cell differentiation, antigen-presenting capacity and tissue remodeling were localized in both organs.

Characterization and expression of NADPH oxidase in LPS-, poly(I:C)- and zymosan-stimulated trout (Oncorhynchus mykiss W.) macrophages.

In vertebrates, the generation of superoxide reactive oxygen species (ROS) via activation of the Nox/Duox family of NADPH oxidases is a prototypical feature of the pathogen-induced defensive responses of activated professional phagocytes. To understand the role of the rainbow trout (Oncorhynchus mykiss) Phox oxidase from a phylogenetic and functional perspective we describe the cloning, sequencing and expression analysis of multiple NADPH components in cultured macrophages. Phylogenetic analyses support the notion of the emergence of Phox-related components before the diversification of basal euteleosts and add to the limited collection of teleost NADPH oxidases. Expression studies using lipopolysaccharide, polyinosine-polycytidylic acid and zymosan to mimic the onset of inflammatory responses in trout macrophages suggest differences in regulation of the NADPH complex throughout the maturation/differentiation period of culture and between different treatments.

Molecular cloning and characterization of European seabass (Dicentrarchus labrax) and Gilthead seabream (Sparus aurata) complement component C3

We present the complete C3 cDNA sequence of Gilthead seabream (Sparus aurata) and European seabass (Dicentrarchus labrax) and its molecular characterization with a descriptive analysis of their structural elements. We obtained one sequence for Gilthead seabream (gsbC3) which encodes a predicted protein of 1656 amino acids, and two sequences for European seabass (esbC3_1 and esbC3_2) which encode two predicted proteins of 1654 and 1587 amino acids respectively. All sequences present the characteristic structural features of C3 but interestingly esbC3_2 lacks the anaphylotoxin domain and the cysteine residue responsible for thiolester bond formation. Moreover, we have detected and quantified (by real-time PCR-based absolute quantification) specific isoform expression in European seabass depending on pathogen and density conditions in vivo. In addition, we have analyzed the tissue distribution pattern of European seabass and Gilthead seabream C3 genes under crowding stress and under pathological challenges in vivo, and we have observed that crowding and infection status provoke changes in expression levels, tissue expression pattern and C3 isoform expression balance.

Comparative Immune- and Stress-Related Transcript Response Induced by Air Exposure and Vibrio anguillarum Bacterin in Rainbow Trout (Oncorhynchus mykiss) and Gilthead Seabream (Sparus aurata) Mucosal Surfaces

Fish have to face various environmental challenges that may compromise the efficacy of the immune response in mucosal surfaces. Since the effect of acute stress on mucosal barriers in fish has still not been fully elucidated, we aimed to compare the short-term mucosal stress and immune transcriptomic responses in a freshwater (rainbow trout, Oncorhynchus mykiss) and a marine fish (gilthead seabream, Sparus aurata) to bacterial immersion (Vibrio anguillarum bacterin vaccine) and air exposure stress in skin, gills, and intestine. Air exposure and combined (vaccine + air) stressors exposure were found to be inducers of the cortisol secretion in plasma and skin mucus on both species in a time-dependent manner, while V. anguillarum bacterin exposure induced cortisol release in trout skin mucus only. This was coincident with a marked differential increase in transcriptomic patterns of stress- and immune-related gene expression profiles. Particularly in seabream skin, the expression of cytokines was markedly enhanced, whereas in gills the response was mainly suppressed. In rainbow trout gut, both air exposure and vaccine stimulated the transcriptomic response, whereas in seabream, stress and immune responses were mainly induced by air exposure. Therefore, our comparative survey on the transcriptomic mucosal responses demonstrates that skin and gut were generally more reactive in both species. However, the upregulation of immune transcripts was more pronounced in gills and gut of vaccinated trout, whereas seabream appeared to be more stress-prone and less responsive to V. anguillarum bacterin in gills and gut. When fish were subjected to both treatments no definite pattern was observed. Overall, the results indicate that (1) the immune response was not homogeneous among mucosae (2), it was greatly influenced by the specific traits of each stressor in each surface and (3) was highly species-specific, probably as a result of the adaptive life story of each species to the microbial load and environmental characteristics of their respective natural habitats.

Functional evidence for the inflammatory reflex in teleosts: A novel α7 nicotinic acetylcholine receptor modulates the macrophage response to dsRNA

&NA; The inflammatory reflex modulates the innate immune system, keeping in check the detrimental consequences of overstimulation. A key player controlling the inflammatory reflex is the alpha 7 acetylcholine receptor (&agr;7nAChR). This receptor is one of the signalling molecules regulating cytokine expression in macrophages. In this study, we characterize a novel teleost &agr;7nAChR. Protein sequence analysis shows a high degree of conservation with mammalian orthologs and trout &agr;7nAChR has all the features and essential amino acids to form a fully functional receptor. We demonstrate that trout macrophages can bind &agr;‐bungarotoxin (&agr;‐BTX), a competitive antagonist for &agr;7nAChRs. Moreover, nicotine stimulation produces a decrease in pro‐inflammatory cytokine expression after stimulation with poly(I:C). These results suggest the presence of a functional &agr;7nAChR in the macrophage plasma membrane. Further, in vivo injection of poly(I:C) induced an increase in serum ACh levels in rainbow trout. Our results manifest for the first time the functional conservation of the inflammatory reflex in teleosts.