Geert F. Wiegertjes

IMMUNOGENETICS OF DISEASE RESISTANCE IN FISH : A COMPARATIVE APPROACH

The study of the genetic regulation of infectious disease resistance depends on the availability of inbred lines or selection lines of the species under investigation. The small numbers of such lines of fish has limited the strategy in teleosts to studies of associations between disease and immune/health traits. Attempts to correlate genetic differences in immune responsiveness with survival after experimental challenge with pathogenic bacteria have failed to define immune parameters that can substantially aid selection for genetic resistance to infectious diseases. Advantages and disadvantages of selection strategies as illustrated by mouse and chicken models are discussed. In this study we summarize the present situation in fish as well as our attempts to develop gynogenetic lines of carp for immunogenetic research.

Molecular and functional characterization of a fish inducible-type nitric oxide synthase

Abstract Using an oligonucleotide primer based on a partial goldfish inducible nitric oxide synthase (iNOS) sequence, a complete carp iNOS cDNA was isolated from an activated carp phagocyte cDNA library. Nucleotide and predicted amino acid sequence analysis indicate that carp iNOS encodes a 1127-amino acid protein with 57% sequence identity to human iNOS. Like mammalian NOSs, carp iNOS protein contains putative binding sites for heme, tetrahydrobiopterin, calmodulin, flavine mononucleotide, flavine adenine dinucleotide, and NADPH. Phylogenetic analysis, using neighbor joining, showed that the carp iNOS protein clustered together with the other vertebrate iNOS proteins. Inducibility of carp iNOS was confirmed by reverse transcription-polymerase chain reaction after stimulation of carp phagocytes with lipopolysaccharide or the protozoan blood flagellate Trypanoplasma borreli. These stimulators produced high amounts of nitric oxide that were toxic for T. borreli in vitro. The nuclear transciption factor NF-κB was shown to play a role in the induction of iNOS transcription.

Ligand specificities of Toll-like receptors in fish: Indications from infection studies

Toll like receptors (TLRs) are present in many different fish families from several different orders, including cyprinid, salmonid, perciform, pleuronectiform and gadiform representatives, with at least some conserved properties among these species. However, low conservation of the leucine-rich repeat ectodomain hinders predictions of ligand specificities of fish TLRs based on sequence information only. We review the presence of a TLR genes, and changes in their gene expression profiles as result of infection, in the context of different fish orders and fish families. The application of RT-qPCR and availability of increasing numbers of fish genomes has led to numerous gene expression studies, including studies on TLR gene expression, providing the most complete dataset to date. Induced changes of gene expression may provide (in)direct evidence for the involvement of a particular TLR in the reaction to a pathogen. Especially when findings are consistent across different studies on the same fish species or consistent across different fish species, up-regulation of TLR gene expression could be a first indication of functional relevance. We discuss TLR1, TLR2, TLR4, TLR5 and TLR9 as presumed sensors of bacterial ligands and discuss as presumed sensors of viral ligands TLR3 and TLR22, TLR7 and TLR8. More functional studies are needed before conclusions on ligands specific to (groups of) fish TLRs can be drawn, certainly true for studies on non-mammalian TLRs. Future studies on the conservation of function of accessory molecules, in conjunction with TLR molecules, may bring new insight into the function of fish TLRs.

Differential expression of two interferon-gamma genes in common carp (Cyprinus carpio L.).

Two interferon gamma (IFN-gamma) genes are expressed in immune cells of teleost fish and are potentially implicated in B- and T-lymphocyte responses. IFN-gamma-2 shows structural and functional characteristics to other vertebrate IFN-gamma genes and is associated with T-lymphocyte function. Expression profiling shows IFN-gamma-2 upregulation in T-lymphocytes after phytohemagglutinin (PHA) stimulation in vitro. Unexpectedly, we found IFN-gamma-1, which is structurally different from IFN-gamma-2, to be expressed in lipopolysacharide (LPS)-stimulated IgM+ (B- lymphocyte enriched) fractions. Expression of T-box transcription factor T-bet, but not of GATA-binding protein 3 (GATA3), correlated with expression of both IFN-gamma genes. In-vivo parasite infection, but as predicted not zymosan-induced inflammation, resulted in concomitant upregulation of T-bet and IFN-gamma-2. This corroborates a genuine T-lymphocyte associated role for IFN-gamma-2.

The immune response of carp to Trypanoplasma borreli: kinetics of immune gene expression and polyclonal lymphocyte activation

Although Trypanoplasma borreli induces the production of non-specific antibodies, survival of infection is associated with the production of T. borreli specific antibodies, able to lyse this parasite in the presence of complement. During the lag phase of this acquired immune response, innate immune mechanisms must limit multiplication of T. borreli. A heat-labile fraction of T. borreli together with CpG motifs in the DNA of this parasite are responsible for the induction of nitric oxide (NO) and probably also for the induction of expression of the inflammatory cytokines tumor necrosis factor (TNF)alpha and interleukin (IL)-1beta by carp phagocytes in vitro. In the signal transduction pathway leading to activation of phagocytes, protein tyrosine kinase and protein kinase C are involved and probably collaborate in activation of the transcription factor nuclear factor (NF)-kappaB. In vivo, carp intraperitoneally injected with T. borreli up-regulate expression of TNFalpha, IL-1beta and mRNAs for acute phase response proteins (complement factor 3, serum amyloid A and alpha-2-macroglobulin).

Daily handling stress reduces resistance of carp to Trypanoplasma borreli: in vitro modulatory effects of cortisol on leukocyte function and apoptosis.

Carp subjected to daily handling stress were much more susceptible to Trypanoplasma borreli infection than control fish. In a search for the cellular mechanisms involved, it was observed that cortisol suppressed T. borreli-induced expression of interleukin-1beta, tumor necrosis factor-alpha, serum amyloid A and inducible nitric oxide synthase. An NF-kappaB-inhibitor could replicate cortisol-induced apoptosis of activated peripheral blood leukocytes. In contrast, although this NF-kappaB-inhibitor induced apoptosis of neutrophilic granulocytes, cortisol prevented apoptosis of these cells, suggesting the latter process to be NF-kappaB-independent. Carp leukocytes, upon induction of apoptosis, exhibit a number of sequential metabolic alterations. First, the mitochondrial transmembrane potential (DeltaPsi(m)) is disrupted and glutathione levels are depleted, followed by exposure of phosphatidylserine on the outer cell membrane. In vitro, cortisol could inhibit NO production induced by low concentrations of lipopolysaccharide (LPS), but remarkably, enhanced NO production induced by high concentrations of LPS. However, no differences in NO production were observed in stressed versus non-stressed infected carp.

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.

Immune modulation by fish kinetoplastid parasites: A role for nitric oxide

Trypanoplasma borreli and Trypanosoma carassii are kinetoplastid parasites infecting cyprinid fish. We investigated the role of nitric oxide (NO) in immune modulation during T. borreli and T. carassii infection of carp. Phagocytic cells from different organs produced NO and serum nitrate levels increased, demonstrating that T. borreli activates NO production in vivo. In contrast, T. carassii did not induce NO production in vivo and inhibited LPS-induced NO production in vitro. Production of NO was detrimental to the host as T. borreli-infected carp treated with the inducible NO synthase inhibitor aminoguanidine had a higher survival than infected control carp. This detrimental effect can be explained (in part) by the toxicity of NO to cells in vitro as NO inhibited the proliferative response of blood and spleen leukocytes. Head-kidney phagocytes were resistant to the immunosuppressive effects of NO in vitro. The NO-inducing activity of T. borreli may be an adaptation developed to ensure survival and immune evasion in the fish host. Apparently, T. carassii has adopted another strategy by deactivating specific functions of phagocytes. Both strategies may ensure long-term survival of the parasite.

Molecular cloning and expression of two β-defensin and two mucin genes in common carp (Cyprinus carpio L.) and their up-regulation after β-glucan feeding.

In this study, we described the partial structure, mRNA tissue distribution and regulation of two carp mucin and two β-defensin genes. This is the first description of these genes in fish. The genes might provide relevant tools to monitor feed-related improvements of fish health under aquaculture conditions. Carp mucin 2 and mucin 5B genes show a high similarity to their mammalian and avian counterparts. The carp β-defensin 1 and β-defensin 2 genes cluster together well with their piscine family members. The influence of a β-glucan immunomodulant on the expression of these genes in mucosal tissues could be confirmed for the first time. Muc5B expression was significantly increased in the skin. For Muc2 no significant up- or down-regulation could be observed. Significantly higher expression levels of β-defensin 2 in gills and both β-defensin genes in skin were found. Thus, the mucosal system can be influenced by the addition of β-glucans to the food.

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.