Simon MacKenzie

Endotoxin recognition: In fish or not in fish?

The interaction between pathogens and their multicellular hosts is initiated by activation of pathogen recognition receptors (PRRs). These receptors, that include most notably members of the toll‐like receptor (TLR) family, recognize specific pathogen‐associated molecular patterns (PAMPs). TLR4 is a central part of the receptor complex that is involved in the activation of the immune system by lipopolysaccharide (LPS) through the specific recognition of its endotoxic moiety (Lipid A). This is a critical event that is essential for the immune response to Gram‐negative bacteria as well as the etiology of endotoxic shock. Interestingly, compared to mammals, fish are resistant to endotoxic shock. This in vivo resistance concurs with in vitro studies demonstrating significantly lowered sensitivity of fish leukocytes to LPS activation. Further, our in vitro analyses demonstrate that in trout mononuclear phagocytes, LPS fails to induce antiviral genes, an event that occurs downstream of TLR4 and is required for the development of endotoxic shock. Finally, an in silico approach that includes mining of different piscine genomic and EST databases, reveals the presence in fish of all of the major TLR signaling elements except for the molecules specifically involved in TLR4‐mediated endotoxin recognition and signaling in mammals. Collectively, our analysis questions the existence of TLR4‐mediated cellular responses to LPS in fish. We further speculate that other receptors, in particular beta‐2 integrins, may play a primary role in the activation of piscine leukocytes by LPS.

LPS-stimulated expression of a tumor necrosis factor-alpha mRNA in primary trout monocytes and in vitro differentiated macrophages.

We have characterised the lipopolysaccharide (LPS) induction and stability of a tumor necrosis factor-alpha (TNFalpha) mRNA in both monocytes and mature macrophages cultured from trout head kidney. Macrophages differentiated in vitro significantly change in phenotypic appearance, phagocytic capacity and upon LPS exposure display a greatly increased capacity for trout TNFalpha (tTNF-alpha) mRNA expression over that of freshly isolated monocytes. The half-life of the tTNF-alpha mRNA in macrophages is approximately 40 min suggesting that control mechanisms for labile cytokine mRNAs in teleosts may be similar to that found in mammals. Our results suggest that in fish, terminally differentiated macrophages play a central and key role in TNF production as a response to infection.

Coping strategies in a strongly schooling fish, the common carp Cyprinus carpio.

Individual common carp Cyprinus carpio were screened repeatedly for risk taking (rate of exploration of a novel, potentially dangerous environment) and for competitive ability (success in gaining access to a spatially restricted food source). Marked differences in behaviour were evident, and significant consistency in individual responses across trials was found for both risk taking and competitive ability. In addition, there was a significant positive relationship between individual performance in these two contexts, with fish that explored more quickly in the novel environment tending to be among the first to gain access to restricted food. In two follow-up studies, resting metabolic rate, blood lactate and glucose and the expression of the cortisol receptor gene in the head kidney and brain were compared in fish from the two extremes of the risk-taking spectrum. Mass-specific metabolic rate was significantly higher in risk-taking than in risk-avoiding fish, while plasma lactate and glucose concentrations and expression of the cortisol receptor gene were lower. It was concluded that a behavioural syndrome based on boldness and aggression exists in C. carpio, as it does in many other animals, and that this is associated with differences in metabolic and stress physiology (down to the genomic level) similar to those described in animals with different coping strategies.

A genetic basis for the phenotypic differentiation between siscowet and lean lake trout (Salvelinus namaycush)

In Lake Superior there are three principal forms of lake trout (Salvelinus namaycush): lean, siscowet and humper. Wild lean and siscowet differ in the shape and relative size of the head, size of the fins, location and size of the eyes, caudal peduncle shape and lipid content of the musculature. To investigate the basis for these phenotypic differences, lean and siscowet lake trout, derived from gametes of wild populations in Lake Superior, were reared communally under identical environmental conditions for 2.5 years. Fish were analysed for growth, morphometry and lipid content, and differences in liver transcriptomics were investigated using Roche 454 GS‐FLX pyrosequencing. The results demonstrate that key phenotypic differences between wild lean and siscowet lake trout such as condition factor, morphometry and lipid levels, persist in these two forms when reared in the laboratory under identical environmental conditions. This strongly suggests that these differences are genetic and not a result of environmental plasticity. Transcriptomic analysis involving the comparison of hepatic gene frequencies (RNA‐seq) and expression (quantitative reverse transcription–polymerase chain reaction (qPCR)) between the two lake trout forms, indicated two primary gene groups that were differentially expressed; those involving lipid synthesis, metabolism and transport (acyl‐CoA desaturase, acyl‐CoA binding protein, peroxisome proliferator‐activated receptor gamma, and apolipoproteins), and those involved with immunity (complement component C3, proteasome, FK506 binding protein 5 and C1q proteins). The results demonstrate that RNA‐seq can be used to identify differentially expressed genes; however, some discrepancies between RNA‐seq analysis and qPCR indicate that methods for deep sequencing may need to be refined and/or different RNA‐seq platforms utilized.

Stress-related hormones modulate cytokine expression in the head kidney of gilthead seabream (Sparus aurata)

Neuro-endocrine and immune systems closely interact in fish, and their regulation is crucial for the maintenance of good health of cultured fish. We have used the seabream head kidney to study whether stress-related hormones can modulate the immune response. For this purpose, the effects of adrenaline, adrenocorticotropic hormone (ACTH) and cortisol on the expression of pro-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6) and the anti-inflammatory cytokine TGF-beta1 were determined by means of quantitative real-time PCR on isolated head kidney cells. ACTH (150 ng mL(-1)) caused an acute increase of TNF-alpha and IL-6 mRNA levels as well as an inhibition of IL-1beta expression. The expression of the anti-inflammatory cytokine TGF-beta1 was also increased, although in a lower extent. Adrenaline (1 muM) early effects were only clear inhibiting IL-1beta expression but not TNF-alpha, IL-6 or TGF-beta1 mRNA levels, while a longer exposure to the hormone inhibited all cytokines. Moreover, cortisol (50 and 100 ng mL(-1)) reduced the expression of all cytokines in a dose-dependent manner. Bacterial lipopolysaccharide (LPS) stimulated IL-1beta expression and inhibited that of the anti-inflammatory TGF-beta1, although it was ineffective on TNF-alpha and IL-6. In addition, adrenaline and cortisol decreased the LPS-stimulated IL-1beta expression, further demonstrating their previously reported anti-inflammatory effects. The combination of ACTH and LPS, on the other hand, did not affect LPS-stimulated IL-1beta expression but was effective increasing TNF-alpha expression. Taking all these results in consideration, we conclude that the expression of pro- and anti-inflammatory cytokines in the seabream head kidney is highly influenced by stress-related hormones, thus indicating an important role for the endocrine system in the modulation of the immune response in teleost fish.

Peptidoglycan, not endotoxin, is the key mediator of cytokine gene expression induced in rainbow trout macrophages by crude LPS

In rainbow trout macrophages, phenol-extracted lipopolysaccharide (LPS) preparations stimulate proinflammatory cytokine gene expression but ultrapure preparations of LPS are inactive. Crude LPS preparations could potentially have a number of contaminants including peptidoglycans (PGNs), nucleic acids and lipoproteins. Thus, in the current study we individually tested potentially contaminating pathogen associated molecular patterns (PAMPs) on rainbow trout (Oncorhynchus mykiss) macrophages to determine which ones could induce proinflammatory cytokine expression. We found that PGNs derived from Gram-negative bacteria (Escherichia coli 0111:B4 and K12), are potent inducers of IL-1beta and IL-6 gene expression and were equal to, or more potent than, crude LPS. On the other hand, PGNs of Gram-positive bacteria, DNA, RNA and lipoteichoic acid were weak stimulators, and lipid A, lipoprotein (Pam3CSK4) and ultrapure LPS were nonstimulatory. More importantly, crude LPS treated with lysozyme to degrade PGNs, exhibited greatly reduced activity in stimulating IL-1beta and IL-6 gene expression, indicating that PGNs in the crude LPS are responsible for a significant amount of the proinflammatory activity. Finally, we showed that PGN treatment induces expression of COX-2 and the subsequent synthesis and release of prostaglandin E(2) (PGE(2)), an important mediator of inflammatory processes. The strong stimulatory effect of E. coli PGNs by themselves on trout macrophages suggests that the recognition of Gram-negative bacteria in trout is through PGNs in the bacterial wall, and indicates that the systems responsible for bacterial recognition in invertebrates (e.g., Drosophila) may also be conserved in some vertebrates.

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.

PAMPs, PRRs and the genomics of gram negative bacterial recognition in fish

Understanding the mechanisms that underpin pathogen recognition and subsequent orchestration of the immune response in fish is an area of significant importance for both basic research and management of health in aquaculture. In recent years much attention has been given to the identification of pattern recognition receptors (PRRs) in fish, however, characterisation of interactions with specific pathogen-associated molecular patterns (PAMPs) is still incomplete. Microarray studies have significantly contributed to functional studies and early descriptions of PAMP-PRR driven activation of specific response cassettes in the genome have been obtained although much is left to be done. In this review we will address gram negative (G-negative) bacterial recognition in fish addressing contributing factors such as structure-function relationships between G-negative PAMPs, current knowledge of fish PRRs and the input achieved by microarray-based studies ranging from in vivo infection studies to directed in vitro PAMP-cell studies. Finally we revisit the endotoxic recognition paradigm in fish and suggest a series of future perspectives that could contribute toward the further elucidation of G-negative bacterial recognition across the highly diverse group of vertebrates that encompass the fishes.

Screening for Coping Style Increases the Power of Gene Expression Studies

Background Individuals of many vertebrate species show different stress coping styles and these have a striking influence on how gene expression shifts in response to a variety of challenges. Principal Findings This is clearly illustrated by a study in which common carp displaying behavioural predictors of different coping styles (characterised by a proactive, adrenaline-based or a reactive, cortisol-based response) were subjected to inflammatory challenge and specific gene transcripts measured in individual brains. Proactive and reactive fish differed in baseline gene expression and also showed diametrically opposite responses to the challenge for 80% of the genes investigated. Significance Incorporating coping style as an explanatory variable can account for some the unexplained variation that is common in gene expression studies, can uncover important effects that would otherwise have passed unnoticed and greatly enhances the interpretive value of gene expression data.

The response of fish to immunostimulant diets

In order to maintain fish health and to improve performance immunostimulants have been used as dietary additives to improve weight gain, feed efficiency, and/or disease resistance in cultured fish. In aquaculture, non-specific immunostimulants have been widely used probably due to the limited knowledge of the immune response in fish and the ease of their application. Many studies have been carried out to assess the effect of dietary immunostimulants in fish including algal derivatives, herb and plant extract containing diets using a wide range of downstream analytical techniques. Many immunostimulants are based upon tradition and folklore transferred through generations and specific to certain geographical regions rather than known biological properties. However, there are studies in which it is possible to observe a clear and direct dose-dependent stimulatory effect upon the immune system. Other dietary supplements used contain PAMPs (Pathogen Associated Molecular Patterns) as immunostimulants whose recognition depends upon PRR (pathogen recognition receptor) interactions including the TLRs (Toll-like receptor). Despite the growing interest in the use of immunostimulants across the aquaculture industry the underlying mechanisms of ligand recognition, extract composition and activation of the fish immune response remains fragmented. In this review we focus upon the last 15 years of studies addressing the assessment of: (1) plant, herb and algae extracts; and (2) PAMPs, upon non-specific immune parameters of activation and immunostimulant diet efficacy.