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The interleukin-1-receptor-associated kinase 3 (IRAK3) is known in mammals as a negative feedback regulator of NF-κB-mediated innate-immune mechanisms. Our RNA-seq experiments revealed a prototypic 1920-nt sequence encoding irak3 from rainbow trout ( Oncorhynchus mykiss ), as well as 20 variants that vary in length and nucleotide composition. Based on the DNA-sequence information from two closely related irak3 genes from rainbow trout and an irak3- sequence fragment from Atlantic salmon retrieved from public databases, we elucidated the underlying genetic causes for this striking irak3 diversity. Infecting rainbow trout with a lethal dose of Aeromonas salmonicida enhanced the expression of all variants in the liver, head kidney, and peripheral blood leucocytes. We analyzed the functional impact of the full-length factor and selected structural variants by overexpressing them in mammalian HEK-293 cells. The full-length factor enhanced the basal activity of NF-κB, but did not dampen the TLR2-signaling-induced levels of NF-κB activation. Increasing the basal NF-κB-activity through Irak3 apparently does not involve its C-terminal domain. However, more severely truncated factors had only a minor impact on the activity of NF-κB. The TLR2-mediated stimulation did not alter the spatial distribution of Irak3 inside the cells. In salmonid CHSE-214 cells, we observed that the Irak3-splice variant that prominently expresses the C-terminal domain significantly quenched the stimulation-dependent production of interleukin-1β and interleukin-8, but not the production of other immune regulators. We conclude that the different gene and splice variants of Irak3 from trout play distinct roles in the activation of immune-regulatory mechanisms., (Copyright © 2019 Rebl, Rebl, Verleih, Haupt, Köbis, Goldammer and Seyfert.)

The mammalian interleukin 1 receptor-like 1 receptor (IL1RL1), commonly known as ST2, is thought to downregulate TLR signalling by sequestering the signalling adapter MYD88 (myeloid differentiation primary response protein 88). ST2 sequences are known in several fish species, but none of them have functionally been examined. We characterised ST2 from rainbow trout (Oncorhynchus mykiss) and the structure of its encoding gene. The primary sequence of ST2 is only weakly conserved from fish to human. However, the amino acid sequences forming the interfaces for ST2 and MYD88 interaction are well conserved throughout evolution. High similarity of the gene segmentation unambiguously proves the common ancestry of fish and mammalian ST2. Trout ST2 and trout MYD88 genes were constitutively expressed in embryonic, larval and adult trout. In vivo infection with Aeromonas salmonicida did not modulate the mRNA levels of both factors. Overexpressing trout ST2 in the mammalian HEK-293 reconstitution system of TLR2 signalling quenched the Escherichia coli-induced activation of NF-κB and SAA promoters in a dose-dependent fashion. The expression of GFP-tagged trout ST2 in human HEK-293 or trout CHSE-214 cells surprisingly revealed that (i) ST2 localised abundantly at the nuclear membrane rather than at the cell membrane and (ii) the coexpression of both ST2 and MYD88 allowed the translocation of trout MYD88 from cytoplasm to nucleus, as assessed using confocal microscopy and Western blotting. Hence, we validated that trout ST2 is a dampener of TLR signalling and interacts with MYD88. The spatial distribution of these factors raises questions about how this repressive mechanism functions., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Two 'trout C-polysaccharide-binding proteins,' TCBP1 and -2, with relevance to early inflammatory events have been discovered in the last century. The present study characterises the respective cDNA sequences from rainbow trout (Oncorhynchus mykiss), including multiple TCBP1 transcript variants. These variants are generated either by the use of alternative splice sites or the exclusion of exons. The longest mRNA isoform, TCBP1-1, encodes a 245-aa protein with a large signal peptide and a complement component C1q domain. The shortest mRNA isoform, TCBP1-5, contains a premature termination codon and hence fails to encode a functional factor. The 224-aa-long TCBP2 protein consists of a comparably shorter signal peptide and a pentraxin domain. Evolutionary analyses clearly separated TCBP1 and -2 because of distinctive protein motifs. Expression profiling in the liver, spleen, and head kidney tissues of healthy trout revealed that TCBP2 mRNA concentrations were higher than the concentrations of all five TCBP1 mRNA isoforms together. The hepatic levels of these TCBP1 variants increased significantly upon infection with Aeromonas salmonicida, whereas TCBP2 transcript levels rose moderately. As the biological function of TCBP1 is barely understood, we tagged this factor with the green fluorescent protein and visualised its expression in HEK-293 cells. Overexpression of TCBP1 increased the level of active NF-κB factors and induced cell death, indicating its involvement in proapoptotic NF-κB-dependent signalling routes., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The complement system is one of the most ancient and most essential innate immune cascades throughout the animal kingdom. Survival of aquatic animals, such as rainbow trout, depends on this early inducible, efficient immune cascade. Despite increasing research on genes coding for complement components in bony fish, some complement-related genes are still unknown in salmonid fish. In the present study, we characterize the genes encoding complement factor D (CFD), CD93 molecule (CD93), and C-type lectin domain family 4, member M (CLEC4M) from rainbow trout (Oncorhynchus mykiss). Subsequently, we performed comprehensive and comparative expression analyses of 36 complement genes including CFD, CD93, and CLEC4M and further putative complement-associated genes to obtain general information about the functional gene interaction within the complement pathway in fish. These quantification analyses were conducted in liver, spleen and gills of healthy fish of two rainbow trout strains, selected for survival (strain BORN) and growth (Import strain), respectively. The present expression study clearly confirms for rainbow trout that liver represents the primary site of complement expression. Spleen and gills also express most complement genes, although the mean transcript levels were generally lower than in liver. The transcription data suggest a contribution of spleen and gills to complement activity. The comparison of the two rainbow trout strains revealed a generally similar complement gene expression. However, a significantly lower expression of numerous genes especially in spleen seems characteristic for the BORN strain. This suggests a strain-specific complement pathway regulation under the selected rearing conditions., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The fish gills represent a crucial organ for the communication with the aquatic environment. Transcriptional changes in gills of two hatchery rainbow trout strains in response to injection with the potent pathogen Aeromonas salmonicida were detected by global gene expression profiling using a 4×44K oligonucleotide microarray. Emphasis was placed on "day 3 postinfection" representing a decisive time point for the resolution of inflammation. The comparison of features and pathways differentially regulated in branchial tissues revealed that the local breeding strain BORN and imported American rainbow trout apply common and specific immune strategies. In gills of infected BORN trout, we observed a dynamic regulation of genes controlling NF-κB pathways and the induction of factors promoting the development of myeloid cells, whereas an increased expression of lysozyme and immunoglobulin genes was obvious in gills of infected import trout. In order to prove the relevance of the array-predicted candidates as well as well-known immune genes for gill immunity, a subsequent in vitro experiment was conducted. Altogether, we uncovered dynamic but moderate changes in the expression of a broad range of immune-relevant features implying the gill's involvement in pathogen defense strategies.

Thermal stress can pose a major challenge to salmonid fish. A 4x44K oligonucleotide microarray approach was used to screen for genetically determined variations of a temperature stress response during acclimation in fish gills, a highly specialized and complex organ responsible for gas and electrolyte exchange as well as excretion. The comparison addressed transcriptional changes in the local breeding strain BORN and imported (TCO) rainbow trout after graded 2-week acclimation to 8 and 23 °C. Besides well-characterized mediators of thermoregulation such as genes encoding cold-inducible RNA-binding protein and heat shock proteins, the present microarray study suggests several new candidate genes commonly regulated in gills of the two trout lines. Having identified the differential expression of thermoregulated genes as duplicated paralogues, they were subsequently validated in a gill cell model. Moreover, the comparison of transcriptome profiles provides evidence for distinctively employed expression patterns. The induction of genes encoding factors of the early innate immunity in BORN trout upon warming contrasts with the increased expression of adaptive immune genes in import trout. Cold acclimation induced genes assigned to the functional categories "cell death" and "ion channel activity" in import trout, but repressed "lipid metabolism." This manuscript provides an overview of the genes of the multifunctional gills in rainbow trout that are mandated after temperature change, suggesting links between the different temperature-dependent pathways and gene networks.

The spleen plays a crucial role in innate and adaptive immunity in bony fish. Consequently, this organ is well suited to assess the immune competence of an organism and hence provides useful information for comparison and classification of production traits of food fish, such as robustness and susceptibility. To gain information about differences in the basal splenic transcriptome activity, healthy rainbow trout of the commercially important strain TCO and the local selection strain BORN have been compared in a holistic expression analysis using the GRASP 16K cDNA microarray. Nearly all differentially expressed genes (n = 807) in the spleen were on a lower expression level (n = 802) in BORN trout compared to the TCO strain. Global gene ontology analysis revealed that most genes are involved in fundamental biological processes like cellular growth, vesicular trafficking and energy metabolism. Surprisingly, only 7 % of splenic differentially expressed genes are associated with functions of the immune system like TLR signaling, acute phase response and complement system. MARCH3 is one lower expressed gene of interest in BORN trout. This gene, coding for an E3 ubiquitin ligase, is involved in three metabolic functions: immune system, vesicular trafficking and ubiquitination. Since MARCH genes are furthermore differently regulated in the two strains after viral infection and assumed to be potentially active in regulation of immune receptors in fish, MARCH3 was chosen for a closer structural analysis. In concert with the data interpretation of the achieved comparative transcriptome analysis for the involved rainbow trout strains, we provide the full mRNA sequence of trout MARCH3 and its hypothetical protein structure.

The iron-sulfur cluster protein ISCU is a scaffold protein tasked with the building and mediation of iron-sulfur [Fe-S]-clusters. These are crucial for [Fe-S]-enzymes, which are involved in essential biological cell processes like metabolism or ion transport. Analysis of ISCU in rainbow trout (Oncorhynchus mykiss) and maraena whitefish (Coregonus maraena) revealed the existence of two gene variants in each of the two salmonids. This study presents the characterization of the duplicated ISCU cDNA sequences in both species as well as the comparative functional analysis of the genes in healthy and affected fish of two rainbow trout strains differing in trait robustness under regional aquaculture conditions. Coding sequences of trout ISCUA and ISCUB genes are spanning over five exons. Open reading frames (ORF) of trout (ISCUA: 495bp, ISCUB: 498bp) and whitefish (ISCUA and ISCUB: 495bp) genes encode for evolutionary highly conserved proteins and share 72% sequence similarity with human ISCU. Transcriptome analyses comparing healthy fish of the local rainbow trout strain BORN and the import strain TCO revealed strain-specific expression patterns for ISCU. Expression analyses by quantitative RT-PCR indicated remarkable differences between the transcript level of the gene variants ISCUA and ISCUB. Moderate temperature challenge (8°C and 23°C) suggests a generally higher transcript level of the two gene variants at 8°C in the liver, spleen, and gill of both strains. However, no remarkable differences between the strains occurred in the temperature-dependent ISCU gene expression profiles. The experimental infection with Aeromonas salmonicida resulted in a different ISCU gene expression in the gill and trunk kidney of both strains after two weeks, suggesting a specific role of the scaffold gene in rainbow trout strain BORN, regarding the recovery after infection. Although results partially reflect the expected strain- and tissue-specific ISCUA and ISCUB regulation in rainbow trout, the data do not support the assumed association of ISCU with the trait robustness., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Ubiquitination regulates the activity, stability, and localization of a wide variety of proteins. Several mammalian MARCH ubiquitin E3 ligase proteins have been suggested to control cell surface immunoreceptors. The mitochondrial protein MARCH5 is a positive regulator of Toll-like receptor 7-mediated NF-κB activation in mammals. In the present study, duplicated MARCH5-like cDNA sequences were isolated from rainbow trout (Oncorhynchus mykiss) comprising open reading frames of 882 bp (MARCH5A) and 885 bp (MARCH5B), respectively. Trout MARCH5A and MARCH5B-encoding sequences share only 65% sequence identity. Phylogenetic analyses including an additionally isolated MARCH5-like sequence from whitefish (Coregonus maraena) suggest that teleosts possess an additional MARCH5 gene copy resulting from a fish-specific whole genome duplication. Coding sequences of MARCH5A and MARCH5B genes from trout are distributed over six exons. Hypothetical MARCH5 proteins from trout comprise four transmembrane helices and a single motif similar to a RING variant domain (RINGv) including eight highly conserved cysteine and histidine residues. A 'reverse-northern blot' analysis revealed furthermore a MARCH5B Δexon5 transcript variant. Both MARCH5 genes from trout show a strain-, tissue- and cell-specific expression profile indicating different functional roles. Fish-specific MARCH5A gene for instance might be involved in defense mechanisms, since in vivo-challenge with the viral pathogen VHSV caused a significant 1.7-fold elevated copy number of the respective gene in gills four days after infection, whereas MARCH5B transcript level did not increase., (Copyright © 2011 Elsevier Ltd. All rights reserved.)