Trudi Hermsen

Unique haplotypes of co-segregating major histocompatibility class II A and class II B alleles in Atlantic salmon (Salmo salar) give rise to diverse class II genotypes

Abstract. Sequence-based typing of a breeding population (G1) consisting of 84 Atlantic salmon individuals revealed the presence of 7 Sasa-DAA and 7 Sasa-DAB expressed alleles. Subsequent typing of 1,182 individuals belonging to 33 families showed that Sasa-DAA and Sasa-DAB segregate as haplotypes. In total seven unique haplotypes were established, with frequencies in the population studied ranging from 0.01 to 0.49. Each haplotype is characterized by a unique minisatellite marker size embedded in the 3′ untranslated region of the Sasa-DAA gene. These data corroborate the fact that Atlantic salmon express a single class II locus, consisting of tightly linked class II A and class B genes. The seven haplotypes give rise to 15 genotypes with frequencies varying between 0.01 and 0.23; 21 class II homozygous individuals were present in the G1 population. We also studied the frequency distribution in another breeding population (G4, n=374) using the minisatellite marker. Only one new marker size was present, suggesting the presence of one new class II haplotype. The marker frequency distribution in the G4 population differed markedly from the G1 population. The genomic organizations of two Sasa-DAA and Sasa-DAB alleles were determined, and supported the notion that these alleles belong to the same locus. In contrast to other studies of salmonid class II sequences, phylogenetic analyses of brown trout and Atlantic class II A and class II B sequences provided support for trans-species polymorphism.

Head Kidney-Derived Macrophages of Common Carp (Cyprinus carpio L.) Show Plasticity and Functional Polarization upon Differential Stimulation

Cells from the myeloid lineage are pluripotent. To investigate the potential of myeloid cell polarization in a primitive vertebrate species, we phenotypically and functionally characterized myeloid cells of common carp (Cyprinus carpio L.) during culture. Flow cytometric analysis, Ab labeling of cell surface markers, and light microscopy showed the presence of a major population of heterogeneous macrophages after culture. These head kidney-derived macrophages can be considered the fish equivalent of bone marrow-derived macrophages and show the ability to phagocytose, produce radicals, and polarize into innate activated or alternatively activated macrophages. Macrophage polarization was based on differential activity of inducible NO synthase and arginase for innate and alternative activation, respectively. Correspondingly, gene expression profiling after stimulation with LPS or cAMP showed differential expression for most of the immune genes presently described for carp. The recently described novel Ig-like transcript 1 (NILT1) and the CXCR1 and CXCR2 chemokine receptors were up-regulated after stimulation with cAMP, an inducer of alternative activation in carp macrophages. Up-regulation of NILT1 was also seen during the later phase of a Trypanosoma carassii infection, where macrophages are primarily alternatively activated. However, NILT1 could not be up-regulated during a Trypanoplasma borreli infection, a model for innate activation. Our data suggest that NILT1, CXCR1, and CXCR2 could be considered markers for alternatively activated macrophages in fish.

The major histocompatibility class II alpha chain in salmonid fishes.

In this study the characterisation of the Atlantic salmon (MhcSasa-DAA) and rainbow trout (MhcOnmy-DAA) class II alpha chain cDNA sequences is presented. The DAA sequences from these two salmonid species showed a high degree of similarity, although the Onmy-DAA(*)03 cDNA sequence differed in the cytoplasmic region. Interestingly, the Onmy-DAA(*)02 sequence has lost the second cysteine in the alpha-1 domain. However, another cysteine is present in this sequence 7 positions downstream of the cysteine which is substituted for a leucine. Despite a thorough search, only a single locus of expressed class II alpha chain sequences was identified in both salmonid species. Amplification by PCR and sequencing of the alpha-1 domain from genomic DNA of three Atlantic salmon, identified four different variants assumed to have derived from this single locus. Two of these variants originated from one individual and are likely functional alleles.

Evolution of Recognition of Ligands from Gram-Positive Bacteria: Similarities and Differences in the TLR2-Mediated Response between Mammalian Vertebrates and Teleost Fish

We investigated the role of the TLR2 receptor in the recognition of ligands from Gram-positive bacteria in fish. Comparative sequence analysis showed a highly conserved Toll/IL-1 receptor domain. Although the leucine-rich repeat domain was less conserved, the position of the critical peptidoglycan (PGN)-binding residues in the leucine-rich repeat domain of carp TLR2 were conserved. Transfection of human embryonic kidney 293 cells with TLR2 corroborated the ability of carp TLR2 to bind the prototypical mammalian vertebrate TLR2 ligands lipoteichoic acid (LTA) and PGN from Staphylococcus aureus. The synthethic triacylated lipopeptide N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-(R)-Cys-(S)-Ser-(S)-Lys4 trihydrochloride (Pam3CSK4) but not the diacylated lipopeptide macrophage-activating lipopeptide-2 (MALP-2) also activated TLR2 transfected human cells. We identified clear differences between the mammalian vertebrates and carp TLR2-mediated response. The use of the same ligands on carp macrophages indicated that fish cells require high concentrations of ligands from Gram-positive bacteria (LTA, PGN) for activation and signal transduction, react less strongly (Pam3CSK4) or do not react at all (MALP-2). Overexpression of TLR2 in carp macrophages confirmed TLR2 reactivity of the response to LTA and PGN, low-responsiveness to Pam3CSK4 and nonresponsiveness to MALP-2. A putative relation with the apparent absence of accessory proteins such as CD14 from the fish TLR2-containing receptor complex is discussed. Moreover, activation of carp macrophages by PGN resulted in increased TLR2 gene expression and enhanced TLR2 mRNA stability, MAPK-p38 phosphorylation and increased radical production. Finally, we could show that NADPH oxidase-derived radicals and MAPK-p38 activation cooperatively determine the level of PGN-induced TLR2 gene expression. We propose that the H2O2-MAPK-p38–dependent axis is crucial for regulation of TLR2 gene expression in fish macrophages.

A novel functional class I lineage in zebrafish (Danio rerio), carp (Cyprinus carpio), and large barbus (Barbus intermedius) showing an unusual conservation of the peptide binding domains.

Species from all major jawed vertebrate taxa possess linked polymorphic class I and II genes located in an MHC. The bony fish are exceptional with class I and II genes located on different linkage groups. Zebrafish (Danio rerio), common carp (Cyprinus carpio), and barbus (Barbus intermedius) represent highly divergent cyprinid genera. The genera Danio and Cyprinus diverged 50 million years ago, while Cyprinus and Barbus separated 30 million years ago. In this study, we report the first complete protein-coding class I ZE lineage cDNA sequences with high similarity between the three cyprinid species. Two unique complete protein-coding cDNA sequences were isolated in zebrafish, Dare-ZE*0101 and Dare-ZE*0102, one in common carp, Cyca-ZE*0101, and six in barbus, Bain-ZE*0101, Bain-ZE*0102, Bain-ZE*0201, Bain-ZE*0301, Bain-ZE*0401, and Bain-ZE*0402. Deduced amino acid sequences indicate that these sequences encode bonafide class I proteins. In addition, the presence of conserved potential peptide anchoring residues, exon-intron organization, ubiquitous expression, and polymorphism generated by positive selection on putative peptide binding residues support a classical nature of class I ZE lineage genes. Phylogenetic analyses revealed clustering of the ZE lineage clade with nonclassical cyprinid class I Z lineage clade away from classical cyprinid class I genes, suggesting a common ancestor of these nonclassical genes as observed for mammalian class I genes. Data strongly support the classical nature of these ZE lineage genes that evolved in a trans-species fashion with lineages being maintained for up to 100 million years as estimated by divergence time calculations.

Common carp have two subclasses of bonyfish specific antibody IgZ showing differential expression in response to infection

Immunoglobulin heavy chains identified in bony fish are broadly classified into three classes namely IgM, IgD and IgZ. The most recently described isotype is IgZ, a teleosts-fish specific isotype that shows variations in gene structure across teleosts. In this study we have identified two IgZ subclasses in common carp. IgZ1 is a four constant heavy chain domains containing antibody isolated across teleosts and IgZ2 is a two constant domains containing heavy chain chimera with a μ1 and ζ4 domain. Sequence analyses suggest that these subtypes are expressed from two separate genomic loci. Expression analyses show that IgZ1 is more abundant in systemic organs and IgZ2 chimera is preferentially expressed at mucosal sites. The basal expression level of IgM in fish is much higher than of the other isotypes. We show that IgZ1 expression in systemic and mucosal organs is responsive to blood parasites, while mucosal parasite infection induces IgM and IgZ2 gene expression. This report is the first to show differential expression of the IgZ variants in response to pathogens and suggests that the IgZ subtypes in carps may have mutually exclusive humoral functions.

Novel immunoglobulin-like transcripts in teleost fish encode polymorphic receptors with cytoplasmic ITAM or ITIM and a new structural Ig domain similar to the natural cytotoxicity receptor NKp44.

Members of the immunoglobulin superfamily (IgSF) include a group of innate immune receptors located in the leukocyte receptor complex (LRC) and other small clusters such as the TREM/NKp44 cluster. These receptors are characterised by the presence of immunoglobulin domains, a stalk, a transmembrane domain, and a cytoplasmic region containing either an immunoreceptor tyrosine-based inhibitory motif (ITIM) or are linked to an adapter molecule with an activation motif (ITAM) for downstream signalling. We have isolated two carp cDNA sequences encoding receptors in which the extracellular Ig domain structurally resembles the novel V-type Ig domain of NKp44. This is supported by a homology model. The cytoplasmic regions contain either an ITAM (Cyca-NILT1) or ITIMs (Cyca-NILT2). The tissue expression of these receptors is nearly identical, with the highest expression in the immunological organs. Peripheral blood leucocytes showed no detectable expression, but upon in vitro culture expressed NILT1, the activating receptor, and not the inhibitory NILT2 receptor. Southern blot analysis indicated that the NILT1 and NILT2 sequences belong to a multigene family. Analysis of the NILT Ig domain-encoding sequences amplified from both genomic DNA and cDNA revealed extensive haplotypic and allelic polymorphism. Database mining of the zebrafish genome identified several homologs on Chromosome 1, which also contains a cluster of class I major histocompatibility genes. This constellation is reminiscent of the TREM/NKp44 gene cluster and the HLA complex located on human Chromosome 6. The carp NILT genes form a unique cluster of innate immune receptors, which are highly polymorphic, and characterised by a new Ig structural subfamily and are distinct from the novel immune-type receptors (Nitrs) found in other fish species.

Detection of MHC class II transcripts in lymphoid tissues of the common carp (Cyprinus carpio L.)

In all vertebrates studied to date, the expression of MHC class II genes is known to be restricted to a limited number of tissues and cell types. In order to have a better understanding of the function of the equivalent genes in teleost fish, the distribution of MHC class II beta transcripts (Cyca-DAB) in the common carp (Cyprinus carpio L.) was investigated. RNA was isolated from tissues and leucocytes, cDNA was produced, and amplification of the Cyca-DAB genes was carried out by PCR. Of the organs with known immunological function, the highest level of Cyca-DAB transcription was found in the thymus. Despite their expected different cellular organization, total blood, head kidney, spleen and the second segment of the gut had similar Cyca-DAB expression levels. No class II transcripts were detected in the skeletal muscle. The studies carried out with leucocytes isolated from the lymphoid tissues point to a direct correlation between the levels of expression and the amounts of surface immunoglobulin positive (sIg+) cells present in the different cell fractions. However, thymus leucocytes did not follow this correlation since the highest level of class II expression was found in a thymocyte fraction that contained very low numbers of Ig+ cells. In PBL the Ig+ cells were highly positive whereas the Ig- were weakly positive. Adherent leucocytes shown to be class II positive, although adherent cells from PBL show a lower level of expression compared to those from the spleen and head kidney.

Cloning of opioid receptors in common carp (Cyprinus carpio L.) and their involvement in regulation of stress and immune response.

In mammals opiate alkaloids and endogenous opioid peptides exert their physiological and pharmacological actions through opioid receptors (MOR, DOR and KOR) expressed not only on neuroendocrine cells but also on leukocytes. Therefore, opioids can modulate the immune response. We cloned and sequenced all three classical opioid receptors (MOR, DOR and KOR) in common carp, and studied changes in their expression during stress and immune responses. Messenger RNA of opioid receptors was constitutively expressed in brain areas, specially in the preoptic nucleus NPO (homologous to mammalian hypothalamus). After exposure to prolonged restraint stress, mRNA levels of MOR and DOR decreased in the NPO and in the head kidney. Increased expression of all studied opioid receptors was observed in the pituitary pars distalis (containing ACTH-producing cells). In immune organs, constitutive but lower expression of opioid receptor genes was observed. During in vivo zymosan-induced peritonitis or after in vitro LPS-induced stimulation, when pro-inflammatory functions are activated, expression of the OR genes in leukocytes was concomitantly up-regulated. Additionally, specific agonists of opioid receptors especially reduced leukocyte migratory properties, manifested by reduced chemotaxis and down-regulated expression of chemokine receptors. Our data indicate an evolutionary conserved role for the opioid system in maintaining a dynamic equilibrium while coping with stress and/or infection.

Major histocompatibility genes in the Lake Tana African large barb species flock: evidence for complete partitioning of class II B, but not class I, genes among different species

The 16 African ‘large’ barb fish species of Lake Tana inhabit different ecological niches, exploit different food webs and have different temporal and spatial spawning patterns within the lake. This unique fish species flock is thought to be the result of adaptive radiation within the past 5 million years. Previous analyses of major histocompatibility class II B exon 2 sequences in four Lake Tana African large barb species revealed that these sequences are indeed under selection. No sharing of class II B alleles was observed among the four Lake Tana African large barb species. In this study we analysed the class II B exon 2 sequences of seven additional Lake Tana African large barb species and African large barbs from the Blue Nile and its tributaries. In addition, the presence and variability of major histocompatibility complex class I UA exon 3 sequences in six Lake Tana and Blue Nile African large barb species was analysed. Phylogenetic lineages are maintained by purifying or neutral selection on non-peptide binding regions. Class II B intron 1 and exon 2 sequences were not shared among the different Lake Tana African large barb species or with the riverine barb species. In contrast, identical class I UA exon 3 sequences were found both in the lacustrine and riverine barb species. Our analyses demonstrate complete partitioning of class II B alleles among Lake Tana African large barb species. In contrast, class I alleles remain for the large part shared among species. These different modes of evolution probably reflect the unlinked nature of major histocompatibility genes in teleost fishes.