Frederick W. Goetz

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.

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.

Rapid accumulation of an interleukin 17 homolog transcript in Crassostrea gigas hemocytes following bacterial exposure.

Interleukin-17 (IL-17) gene models have been found in the sequenced genomes of Strongylocentrotus purpuratus and Caenorhabditis elegans. However, there have been no published reports on the empirical cloning and characterization of any interleukin cDNAs in invertebrates. From a Pacific oyster (Crassostrea gigas) hemocyte cDNA library, two clones were obtained that encoded a protein similar to vertebrate IL-17s. The putative oyster IL-17 homolog (CgIL-17) was 27% identical to rainbow trout IL-17D, 21% to human IL-17D and 24% to an IL-17D-like gene model obtained from the annotation of the sea urchin genome. IL-17s from the oyster, sea urchin, trout and human, contained conserved cysteine residues found in all forms of IL-17 in mammals. Injection of bacteria into C. gigas oysters produced a large and rapid elevation in CgIL-17 transcript abundance in hemocytes, suggesting that this is a very early response gene to pathogens that may be responsible for the stimulation of other immune genes in the oyster.

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.

Functionalization impacts the effects of carbon nanotubes on the immune system of rainbow trout, Oncorhynchus mykiss.

Recent studies have demonstrated the potential for manufactured nanomaterials to reach the aquatic environment. There is a need to determine if these materials will have an impact on aquatic species and at what level of exposure. In addition there is a need to develop models to test the potential effects of the multitude of particle types in production on aquatic vertebrates. The purpose of this research was to determine the impact of manufactured nanomaterials on the immune system of an aquatic vertebrate model, the rainbow trout (Oncorhynchus mykiss). We investigated how structure and type of functionalization of manufactured nanomaterials could affect immunotoxicity. To assess immunotoxicity, we used a well-studied trout macrophage primary cell culture system in conjunction with the expression of IL-1β and IFNα for proinflammatory and antiviral gene expression. There was a significant difference among the different carbon nanotube-based nanomaterials in their level of stimulation of IL-1β in macrophage cells and the dose at which they became stimulatory. At concentrations that were sublethal to cells, almost all nanomaterials were stimulatory at some concentration. Single-walled nanotubes and multi-walled nanotubes that were differentially functionalized to be water-soluble, varied in their effects; specifically the concentrations at which they were stimulatory and they were more stimulatory to IL-1β expression compared with unfunctionalized nanotubes. Each functionalized nanotube type caused a dose-dependent response with the lowest exposures (0.05-1.0 μg/ml) having no stimulatory response and at the highest concentrations (5 μg/ml and 10 μg/ml) stimulating a response similar to the positive LPS positive control. Anionic functionalized multi-walled nanotubes and zwitterionic single-walled nanotubes were stimulatory at the lowest dose (0.5 μg/ml). Sodium deoxycholate, often used to suspend nanomaterials, was also tested and was as stimulatory to the immune cells as the nanomaterials. This study is the first report of the effects of nanomaterials on the function of the immune system in a nonmammalian vertebrate. Since the innate immune system is the first to respond to the intrusion of foreign material, analysis of the effects of nanomaterials on cells of the innate immune system should provide valuable information on how these materials are perceived and affect an animal. Ultimately such research will provide the means to determine which nanomaterials are most harmful to aquatic species and how particles may be altered or functionalized to decrease their toxicity.

A Tumor Necrosis Factor Decoy Receptor Homologue Is Up-Regulated in the Brook Trout (Salvelinus fontinalis) Ovary at the Completion of Ovulation

Abstract An up-regulated cDNA fragment was obtained from differential-display polymerase chain reaction of brook trout ovarian tissue stimulated by phorbol-12-myristate-13-acetate (PMA) and calcium ionophore A23187. Using this cDNA as a probe, a full-length cDNA of 2267 base pairs was obtained by screening a library of PMA/A23187-stimulated ovarian cDNA. The mRNA obtained presumably encodes for a 302-amino acid protein showing similarities with several members of the tumor necrosis factor (TNF) receptor superfamily. The protein contains several cysteine-rich domains characteristic of mammalian TNF receptor members and is most similar to human decoy receptor 3 and osteoprotegerin, two soluble decoy TNF receptors. Consequently, this TNF receptor homologue was tentatively named a trout decoy receptor (TDcR). On Northern blots of ovarian tissue, TDcR hybridized with a 2.2-kilobase transcript that was strongly up-regulated under phorbol ester stimulation. TDcR mRNA was localized in granulosa cells and was detected in the ovary during and after natural ovulation. Its expression was up-regulated at the end of ovulation and progressively down-regulated after 48 h postovulation. Among other trout tissues tested, the transcript was present only in the testis. To our knowledge this is the first description of a member of the TNF receptor family from a lower vertebrate and the first report of a decoy-like TNF receptor in the vertebrate ovary.

Behavioural fever is a synergic signal amplifying the innate immune response

Behavioural fever, defined as an acute change in thermal preference driven by pathogen recognition, has been reported in a variety of invertebrates and ectothermic vertebrates. It has been suggested, but so far not confirmed, that such changes in thermal regime favour the immune response and thus promote survival. Here, we show that zebrafish display behavioural fever that acts to promote extensive and highly specific temperature-dependent changes in the brain transcriptome. The observed coupling of the immune response to fever acts at the gene–environment level to promote a robust, highly specific time-dependent anti-viral response that, under viral infection, increases survival. Fish that are not offered a choice of temperatures and that therefore cannot express behavioural fever show decreased survival under viral challenge. This phenomenon provides an underlying explanation for the varied functional responses observed during systemic fever. Given the effects of behavioural fever on survival and the fact that it exists across considerable phylogenetic space, such immunity–environment interactions are likely to be under strong positive selection.