Alexander Rebl

Toll-like receptor signaling in bony fish.

The innate immune system constitutes an efficient defense against invading microbial pathogens. Toll-like receptors (TLRs) eventually alert vertebrates about the presence of pathogens and elicit the immune responses. To date, 17 different TLRs have been identified in more than a dozen different fish species. Numerous studies revealed that specific piscine TLRs share functional properties with their mammalian counterparts. Nevertheless, remarkable distinct features of teleostean Toll-like receptor cascades have been discovered. A soluble TLR5 factor in rainbow trout for example might amplify danger signaling of membrane-bound TLR5 in a positive feed loop. Piscine TLR3 detects viral and additionally bacterial molecular patterns in contrast to mammalian TLR3. Regarding TLR4, the functional spectrum of this teleostean receptor is also different from its mammalian orthologue. While signaling quite similar as the mammalian counterpart in some fish species, it may down-regulate TLR activation in others or was even lost during evolution. The orthologues of human TLR6 and TLR10 are also absent in teleosts. Some piscine TLRs are encoded by duplicated genes, for example salmonid TLR22. TLR22 is found in several fish species but only as a non-functional pseudogene in man. Additional distinct features of the TLR pathway in bony fish suggest its specific optimization for the aquatic environment. This review summarizes studies characterizing TLRs from several teleost species and discusses features of piscine TLR signaling on the background of the respective mammalian knowledge.

Characterization of two key molecules of teleost innate immunity from rainbow trout (Oncorhynchus mykiss): MyD88 and SAA.

Toll-like receptors (TLR) are relevant for piscine innate immunity. TLR activation recruits several downstream factors regulating the expression of immunorelevant genes. We have characterized two key factors of innate immunity from rainbow trout: MyD88 as an adaptor protein interacting directly with TLRs, and serum amyloid A as an effector molecule induced by the activated Toll-like receptor signaling cascade. Both factors share a remarkable high degree of structural conservation with their mammalian orthologs suggesting that innate immune defense mechanisms may also functionally be conserved between fish and mammals.

Salmonid Tollip and MyD88 factors can functionally replace their mammalian orthologues in TLR-mediated trout SAA promoter activation

Many functional details of the piscine Toll-like receptor (TLR) signal-mediated activation of immune defense are still elusive. We used an established reconstitution system of mammalian TLR signaling to examine if this system would allow for pathogen-dependent promoter activation of the serum amyloid A (SAA)-encoding gene from rainbow trout (Oncorhynchus mykiss) and if the key mediators MyD88 and Tollip from trout can functionally substitute for their mammalian orthologues. Cells of the established human embryonic kidney line HEK-293 were transiently co-transfected with vectors expressing bovine TLR2 or TLR4 factors and a reporter gene driven by the promoter of the trout SAA gene. Escherichia coli stimulation increased reporter gene expression more than 3-fold. Deletion series and point mutations identified in the proximal SAA promoter a composite overlapping binding site for NF-κB and CEBP factors as crucial for promoter activation. Overexpression of NF-κB p65, but not of p50 or different members of the CEBP factor family proved this factor as an essential driver for SAA expression. Overexpression of a transdominant-negative mutant of the trout MyD88 factor reduced TLR-mediated SAA promoter activation confirming functional conservation of its TIR domain. Overexpression of the Tollip factor from trout also quenched TLR-mediated NF-κB and TLR4-mediated SAA promoter activation. The MyD88 mutant and Tollip expression studies confirm the functional homology of both piscine factors and their mammalian counterparts. We provide for the first time evidence that also the Tollip-mediated negative loop of TLR signaling may be conserved in non-mammalian organisms.

Transcriptome profiling reveals insight into distinct immune responses to Aeromonas salmonicida in gill of two rainbow trout strains.

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.

Tollip, a negative regulator of TLR-signalling, is encoded by twin genes in salmonid fish

The factor Tollip is known to dampen TLR2- and TLR4-mediated signalling in mammals. No negative regulator of the piscine TLR-signalling cascade has been described so far, albeit a sizable collection of factors contributing to this ancient pathogen-sensing system are known from fish to date. We identified two closely related Tollip-encoding genes in Atlantic salmon (Salmo salar) and the respective ortholog mRNA molecules in rainbow trout (Oncorhynchus mykiss). The salmonid Tollip genes are segmented into 6 exons, similar to the human orthologous gene. The protein-encoding sequences are homologous to >97% among the twin factors and also between the species. Both encoded proteins contain a C2 domain and an ubiquitin system component, which are also characteristic features of the mammalian Tollip factor. We analysed the expression of these genes in trout. Both Tollip-encoding genes are ubiquitously and also equally expressed, as indicated by similar mRNA concentrations of both factors in any one tissue. Tollip expression was found to be up-regulated by viral infection. Our data suggest that the Tollip genes were duplicated before salmon and trout were evolutionary separated. Moreover, pathways dampening the activity of the TLR-cascade may have been conserved from lower vertebrates to mammals since Tollip, as a respective key factor has been highly conserved from fish to human.

Characterization of Dehydrodolichyl diphosphate synthase gene in rainbow trout (Oncorhynchus mykiss).

Dehydrodolichyl diphosphate synthase (DHDDS) catalyzes cis-prenyl chain elongation to produce the polyprenyl backbone of Dolichol, a glycosyl carrier-lipid required for the co-translational modification of various proteins. The resulting glycoproteins play a role in several physiological and pathological processes. This manuscript characterizes the DHDDS-like gene from rainbow trout (Oncorhynchus mykiss) and its tissue-specific mRNA abundance in two different strains. The ubiquitous expression of DHDDS in trout indicates the essential function of the product Dolichol for metabolic processes. The comparison of the deduced amino acid sequence with DHDDS proteins from different vertebrate, invertebrate, and herbal species reveals a high degree of conserved amino acids and protein regions suggesting a common functional relevance. This is the first report of a prenyltransferase homologue from a teleostean species.

Identification of differentially expressed protective genes in liver of two rainbow trout strains

Since 1975, the rainbow trout strain BORN (Germany) has been bred in brackish water from a coastal form imported from Denmark. Accompanying phenotypic monitoring of the adapted BORN trout until now revealed that this selection strain manifested a generally elevated resistance towards high stress and pathogenic challenge including lower susceptibility towards Aeromonas salmonicida infections in comparison to other trout strains in local aqua farms. We focus on the elucidation of both, genetic background and immunological basis for the increased survivorship to infections. A first comparison of gene expression profiles in liver tissue of healthy rainbow trout from the local selection strain BORN and imported trout using a GRASP 16K cDNA microarray revealed six differentially expressed genes evoking pathogen and wounding responses, LEAP2A (encoding for liver-expressed antimicrobial peptide), SERPINA1 (alpha-1 antitrypsin), FTH1 (middle subunit of ferritin), FGL2 (fibroleukin), CLEC4E (macrophage-inducible C-type lectin), and SERPINF2 (alpha-2 antiplasmin). Since the latter gene is not described in salmonid species so far, our first aim was to characterize the respective sequence in rainbow trout. Two trout SERPINF2 genes were identified, which share only 48% identical amino acid residues and a characteristic SERPIN domain. Second, we aimed to analyse the expression of those genes after temperature challenge (8 °C and 23 °C). Only FTH1 was upregulated in BORN and import trout after increase of temperature, while SERPINA1 and FGL2 were only elevated in import trout. Third, the expression of all named genes was analyzed after pathogen challenge with A. salmonicida subsp. salmonicida. As a main finding, we detected a comparably faster regeneration of LEAP2A mRNA abundance in BORN trout following bacterial infection. Ingenuity Pathways Analysis suggested a functional interplay among the mentioned factors and the pro-inflammatory cytokine TNF, whose stronger expression was validated in liver of BORN trout. This data indicate that the examined genes contribute to an improved first barrier against invading pathogens in BORN trout.

Cytogenetic anchoring of radiation hybrid and virtual maps of sheep chromosome X and comparison of X chromosomes in sheep, cattle, and human

A comprehensive physical map was generated for Ovis aries chromosome X (OARX) based on a cytogenomics approach. DNA probes were prepared from bacterial artificial chromosome (BAC) clones from the CHORI-243 sheep library and were assigned to G-banded metaphase spreads via fluorescence in-situ hybridization (FISH). A total of 22 BACs gave a single hybridization signal to the X chromosome and were assigned out of 32 tested. The positioned BACs contained 16 genes and a microsatellite marker which represent new cytogenetically mapped loci in the sheep genome. The gene and microsatellite loci serve to anchor between the existing radiation hybrid (RH) and virtual sheep genome (VSG) maps to the cytogenetic OARX map, whilst the BACs themselves also serve as anchors between the VSG and the cytogenetic maps. An additional 17 links between the RH and cytogenetic maps are provided by BAC end sequence (BES) derived markers that have also been positioned on the RH map. Comparison of the map orders for the cytogenetic, RH, and virtual maps reveals that the orders for the cytogenetic and RH maps are most similar, with only one locus, represented by BAC CH243-330E18, mapping to relatively different positions. Several discrepancies, including an inverted segment are found when comparing both the cytogenetic and RH maps with the virtual map. These discrepancies highlight the value of using physical mapping methods to inform the process of future in silico map construction. A detailed comparative analysis of sheep, human, and cattle mapping data allowed the construction of a comparative map that confirms and expands the knowledge about evolutionary conservation and break points between the X chromosomes of the three mammalian species.

Aeromonas salmonicida Infection Only Moderately Regulates Expression of Factors Contributing to Toll-Like Receptor Signaling but Massively Activates the Cellular and Humoral Branches of Innate Immunity in Rainbow Trout (Oncorhynchus mykiss).

Toll-like receptors (TLRs) are known to detect a defined spectrum of microbial structures. However, the knowledge about the specificity of teleost Tlr factors for distinct pathogens is limited so far. We measured baseline expression profiles of 18 tlr genes and associated signaling factors in four immune-relevant tissues of rainbow trout Oncorhynchus mykiss. Intraperitoneal injection of a lethal dose of Aeromonas salmonicida subsp. salmonicida induced highly increased levels of cytokine mRNAs during a 72-hour postinfection (hpi) period. In contrast, only the fish-specific tlr22a2 and the downstream factor irak1 featured clearly increased transcript levels, while the mRNA concentrations of many other tlr genes decreased. Flow cytometry quantified cell trafficking after infection indicating a dramatic influx of myeloid cells into the peritoneum and a belated low level immigration of lymphoid cells. T and B lymphocytes were differentiated with RT-qPCR revealing that B lymphocytes emigrated from and T lymphocytes immigrated into head kidney. In conclusion, no specific TLR can be singled out as a dominant receptor for A. salmonicida. The recruitment of cellular factors of innate immunity rather than induced expression of pathogen receptors is hence of key importance for mounting a first immune defense against invading A. salmonicida.

The proximal promoter of a novel interleukin-8-encoding gene in rainbow trout (Oncorhynchus mykiss) is strongly induced by CEBPA, but not NF-κB p65

Interleukin-8 (IL8) is an immediate-early chemokine that has been well characterized in several fish species. Ten IL8 gene variants have already been described in rainbow trout, but none of their promoters has structurally been defined or functionally characterized in teleost fish. To uncover key factors regulating IL8 expression, we intended to functionally characterize an IL8 promoter from rainbow trout. Incidentally, we isolated a novel IL8 gene variant (IL8-G). It is structurally highly similar to the other trout IL8 gene variants and its mRNA concentration increased significantly in secondary lymphoid tissues after infecting healthy fish with Aeromonas salmonicida. The proximal promoter sequence of the IL8-G-encoding gene features in close proximity two consensus elements for CEBP attachment. The proximal site overlaps with a NF-κB-binding site. Cotransfection of an IL8-G promoter-driven reporter gene together with vectors expressing various mammalian CEBP or NF-κB factors revealed in human HEK-293 cells that CEBPA and NF-κB p50, but not NF-κB p65 activate this promoter. The stimulatory effect of NF-κB p50 is likely conveyed by synergizing with CEBPA. Deletion or mutation of either the distal or the proximal CEBP-binding site, respectively, caused a significant decrease in IL8-G promoter activation. We confirmed the significance of the CEBPA factor for IL8-G expression by comparing the stimulatory capacity of the trout CEBPA and -B factors, thereby reducing the evolutionary distance in the inter-species expression assays. Similar promoter induction potential and intracellular localization of the mammalian and teleostean CEBPA and -B factors suggests their functional conservation throughout evolution.