Stimulation of the innate immune system of carp: role of Toll-like receptors

Toll-like receptors (TLRs), named after the Toll gene identified in fruit flies, are a family of evolutionary conserved proteins that play a key role in the innate immune system. TLRs are found inside or on the surface of immune cells of virtually all-living animals and recognize integral parts of microbes. Thereby, they are excellent candidate receptors for controlled stimulation of the innate immune system of, for example, fish in aquaculture. β-glucans are microbial compounds routinely added to fish feed for their health-promoting effects. They regulate innate immunity by stimulating fish cells to produce more oxygen and nitrogen radicals but are not recognized by TLRs.Instead, TLRs of cyprinid fish (zebrafish, carp) are stimulated by viral and/or parasitic infection. Although immunostimulation by β-glucans occurs via yet undefined receptors certainly, addition of integral but harmless parts of microbes to fish feed may help controlfish diseases in aquaculture.</p

Time and concentration dependency of MacroGard® induced apoptosis

In previous studies an effect of β-glucan on apoptosis in fish was noted and in this investigation we determine the time and concentration dependency of this effect. Primary cell cultures of pronephric carp cells were incubated for 6, 24, 48 h with various concentrations ranging from 0 to 1000 μg/ml of MacroGard® β-glucan. Apoptosis was monitored via acridine orange staining. Results indicate a clear effect of time and concentration on the induction of apoptosis in vitro, with only concentration ≥500 μg/ml causing significantly higher percentages of apoptotic cells. Apoptosis was detected after 6 h. This concentration dependent effect has to be considered when studying apoptosis in relation to immunostimulation

Dietary b-glucan (MacroGard®) enhances survival of first feeding turbot (Scophthalmus maximus) larvae by altering immunity, metabolism and microbiota

Reflecting the natural biology of mass spawning fish aquaculture production of fish larvae is often hampered by high and unpredictable mortality rates. The present study aimed to enhance larval performance and immunity via the oral administration of an immunomodulator, β-glucan (MacroGard®) in turbot (Scophthalmus maximus). Rotifers (Brachionus plicatilis) were incubated with or without yeast β-1,3/1,6-glucan in form of MacroGard® at a concentration of 0.5 g/L. Rotifers were fed to first feeding turbot larvae once a day. From day 13 dph onwards all tanks were additionally fed untreated Artemia sp. nauplii (1 nauplius ml/L). Daily mortality was monitored and larvae were sampled at 11 and 24 dph for expression of 30 genes, microbiota analysis, trypsin activity and size measurements. Along with the feeding of β-glucan daily mortality was significantly reduced by ca. 15% and an alteration of the larval microbiota was observed. At 11 dph gene expression of trypsin and chymotrypsin was elevated in the MacroGard® fed fish, which resulted in heightened tryptic enzyme activity. No effect on genes encoding antioxidative proteins was observed, whilst the immune response was clearly modulated by β-glucan. At 11 dph complement component c3 was elevated whilst cytokines, antimicrobial peptides, toll like receptor 3 and heat shock protein 70 were not affected. At the later time point (24 dph) an anti-inflammatory effect in form of a down-regulation of hsp 70, tnf-α and il-1β was observed. We conclude that the administration of MacroGard® induced an immunomodulatory response and could be used as an effective measure to increase survival in rearing of turbot

Modulation of Innate and Adaptive Immune Responses by Arabinoxylans

This is the peer reviewed version of the following article: Fadel, A., Plunkett, A., Li, W., & Ashworth, J. J. (2017). Modulation of Innate and Adaptive Immune Responses by Arabinoxylans. Journal of Food Biochemistry, 42(2), e12473. http://doi.org/10.1111/jfbc.12473, which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1111/jfbc.12473/abstract. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-ArchivingHumans are exposed to harmful pathogens and a wide range of noxious substances every day.The immune system reacts to, and destroys, these pathogens and harmful substances. The immunesystem is composed of innate and adaptive immunity, which liaise to protect the host and maintainhealth. Foods, especially cereals, have been reported to modulate the immune response.Arabinoxylans are nonstarch polysaccharides that have been shown to possess immune-modulatory activities. This review article discusses the fundamentals of the immune system andprovides an overview of the immunomodulatory potential of arabinoxylans in conjunction withtheir structural characteristics and proposed similarities with lipopolysaccharide

Ligand specificities of Toll-like receptors in fish: indicatiaons from infection studies

Toll like receptors (TLRs) are present in many different fish families from several different orders, including cyprinid, salmonid, perciform, pleuronectiform and gadiform representatives, with at least some conserved properties among these species. However, low conservation of the leucine-rich repeat ectodomain hinders predictions of ligand specificities of fish TLRs based on sequence information only. We review the presence of a TLR genes, and changes in their gene expression profiles as result of infection, in the context of different fish orders and fish families. The application of RT-qPCR and availability of increasing numbers of fish genomes has led to numerous gene expression studies, including studies on TLR gene expression, providing the most complete dataset to date. Induced changes of gene expression may provide (in)direct evidence for the involvement of a particular TLR in the reaction to a pathogen. Especially when findings are consistent across different studies on the same fish species or consistent across different fish species, up-regulation of TLR gene expression could be a first indication of functional relevance. We discuss TLR1, TLR2, TLR4, TLR5 and TLR9 as presumed sensors of bacterial ligands and discuss as presumed sensors of viral ligands TLR3 and TLR22, TLR7 and TLR8. More functional studies are needed before conclusions on ligands specific to (groups of) fish TLRs can be drawn, certainly true for studies on non-mammalian TLRs. Future studies on the conservation of function of accessory molecules, in conjunction with TLR molecules, may bring new insight into the function of fish TLRs

Accessory molecules for Toll-like receptors in Teleost fish. Identification of TLR4 interactor with leucine-rich repeats (TRIL)

The biosynthesis and activation of Toll-like receptors (TLRs) requires accessory proteins. In mammals, a number of accessory proteins have been characterized, that can be classified based on their function as ligand-recognition and delivery cofactors, chaperones and trafficking proteins. We identified the homologs in teleost fish genomes of mammalian accessory molecules and show their expression in transcriptome data sets. Further, we annotate in detail TLR4 interactor with leucine-rich repeats (tril) in zebrafish (Danio rerio) and in common carp (Cyprinus carpio). In mammals, TRIL is a functional component of the TLR4 complex and is important for TLR3 signaling, and is mainly expressed in the brain. In fish, the Tril molecule has many conserved features of mouse and human TRIL, containing 13 leucine-rich repeat domains, a fibronectin and a transmembrane domain. Zebrafish tril could not be detected in the latest assembly of the zebrafish genome (Zv9) and required manual annotation based on genome and transcriptome shotgun sequencing data sets. Carp tril was found in two copies in the draft genome. Both copies of carp tril are constitutively expressed in several organs, with the highest gene expression in muscle, skin and brain. In carp, the tril gene is expressed at high levels in endothelial cells and thrombocytes. We discuss the implication of the presence of most, but not all, accessory molecules for the biosynthesis and activation of tlr molecules in fish

DNA Accounting: Tallying Genomes to Detect Adulterated Saffron

The EU General Food Law not only aims at ensuring food safety but also to ‘prevent fraudulent or deceptive practices; the adulteration of food; and any other practices which may mislead the consumer’. Especially the partial or complete, deliberate, and intentional substitution of valuable ingredients (e.g., Saffron) for less valuable ones is of concern. Due to the variety of products on the market an approach to detect food adulteration that works well for one species may not be easily applicable to another. Here we present a broadly applicable approach for the detection of substitution of biological materials based on digital PCR. By simultaneously measuring and forecasting the number of genome copies in a sample, fraud is detectable as a discrepancy between these two values. Apart from the choice of target gene, the procedure is identical across all species. It is scalable, rapid, and has a high dynamic range. We provide proof of concept by presenting the analysis of 141 samples of Saffron (Crocus sativus) from across the European market by DNA accounting and the verification of these results by NGS analysis

Carp Il10 has anti-inflammatory activities on phagocytes, promotes proliferation of memory T-cells and regulates B-cell differentiation and antibody secretion

In the current study, we investigated the effects of carp Il10 on phagocytes and lymphocytes. Carp Il10 shares several prototypical inhibitory activities on phagocytes with mammalian IL-10, including deactivation of neutrophils and macrophages, as shown by inhibition of oxygen and nitrogen radical production, as well as reduced expression of proinflammatory genes and mhc genes involved in Ag presentation. Similar to mammalian IL-10, carp Il10 acts through a signaling pathway involving phosphorylation of Stat3, ultimately leading to the early upregulation of socs3 expression. To our knowledge, this is the first study of the effects of Il10 on lymphocytes in fish. Although Il10 did not affect survival and proliferation of T cells from naive animals, it greatly promoted survival and proliferation of T cells in cultures from immunized animals, but only when used in combination with the immunizing Ag. Preliminary gene expression analysis suggests that, under these circumstances, carp Il10 stimulates a subset of CD8+ memory T cells while downregulating CD4+ memory Th1 and Th2 responses. In addition to the regulatory effect on T cells, carp Il10 stimulates proliferation, differentiation, and Ab secretion by IgM+ B cells. Overall, carp Il10 shares several prototypical activities with mammalian IL-10, including downregulation of the inflammatory response of phagocytes, stimulation of proliferation of subsets of memory T lymphocytes, and proliferation, differentiation, and Ab secretion by IgM+ B lymphocytes. To our knowledge, this is the first comprehensive analysis of biological activities of fish Il10 on both phagocytes and lymphocytes showing functional conservation of several properties of Il10