Macrophage polarisation: immune responses of carp against parasites

In the studies described in this thesis we used a natural host-parasite model of two parasites ( Trypanoplasma borreli and Trypanosoma carassii ) infecting common carp ( Cyprinus carpio L.), to obtain more knowledge about the phenomenon of macrophage polarisation in 'the evolutionary older' teleosts and the consequences of differential activation for the individual host.The general aspects of the teleost immune system are very similar to those of the mammalian immune system. Polarisation of macrophages into classically activated macrophages (caMF) or alternatively activated macrophages (aaMF) have been described for mammals, however, not yet for teleosts. These caMF and aaMF are involved in type I and type II immune responses, respectively. NO production is typically upregulated in caMF while arginase is upregulated in aaMF. Therefore, we propose the use of NO production by caMF and arginase activity from aaMF as markers for type I and type II immune responses in teleosts.To study arginase activity (aaMF) we cloned and sequenced the carp arginase 1 and 2 genes and optimised an arginase activity assay. Furthermore, we developed and characterised a carp macrophage culture system. These head kidney-derived macrophages can be considered the fish equivalent of mammalian bone marrow-derived macrophages. Stimulation of these head kidney-derived macrophages with LPS could induce iNOS expression and NO production (read-out for caMF), while stimulation with the intracellular messenger cAMP could induce arginase 2 expression and activity (read-out for aaMF).Macrophage polarisation has been described during many parasitic infections. T. borreli and T. carassii are kinetoplastid parasites infecting carp. The immune response of carp against these two parasites is different with regard to the production of nitrite by macrophages. We hypothesised that infections with these two parasites in carp provide a good model to study macrophage polarisation in teleosts. We infected carp with T. borreli and with T. carassii and followed the NO production and arginase activity in the head kidney leukocytes (HKL) of these carp. The results indicate that T. borreli -infected carp are more prone to increase nitrite production by caMF, while T. carassii -infected carp are more prone to increase arginase activity by aaMF. We also co-infected carp with both parasites simultaneously. Total parasitaemia was lower and survival was higher in co-infected carp. T. borreli -specific antibody levels were higher in co-infected and in T. carassii -infected carp than in T. borreli -infected carp. Our data indicate a protective effect of co-infection with T. carassii on the resistance to T. borreli , possibly mediated via cross-reactive antibodies. In an attempt to identify the antigenic proteins of the parasites we constructed expression libraries of both parasites and screened these libraries with homologous carp immune serum. Whether the antibodies against the identified antigenic proteins (ribosomal proteins, ubiquitin and activated protein kinase C receptor) are protective and cross-reactive,requires further studies.In conclusion, carp macrophages are able to polarise into classically and alternatively activated macrophages. To some extent this polarisation into caMF and aaMF can be induced by infection with T. borrreli and T. carassii , respectively. The level to which this polarisation in response to these parasites influences the production of (protective) antibodies remains to be elucidated

A Novel Soluble Immune-Type Receptor (SITR) in Teleost Fish: Carp SITR Is Involved in the Nitric Oxide-Mediated Response to a Protozoan Parasite

Background- The innate immune system relies upon a wide range of germ-line encoded receptors including a large number of immunoglobulin superfamily (IgSF) receptors. Different Ig-like immune receptor families have been reported in mammals, birds, amphibians and fish. Most innate immune receptors of the IgSF are type I transmembrane proteins containing one or more extracellular Ig-like domains and their regulation of effector functions is mediated intracellularly by distinct stimulatory or inhibitory pathways. Methodology/Principal Findings - Carp SITR was found in a substracted cDNA repertoire from carp macrophages, enriched for genes up-regulated in response to the protozoan parasite Trypanoplasma borreli. Carp SITR is a type I protein with two extracellular Ig domains in a unique organisation of a N-proximal V/C2 (or I-) type and a C-proximal V-type Ig domain, devoid of a transmembrane domain or any intracytoplasmic signalling motif. The carp SITR C-proximal V-type Ig domain, in particular, has a close sequence similarity and conserved structural characteristics to the mammalian CD300 molecules. By generating an anti-SITR antibody we could show that SITR protein expression was restricted to cells of the myeloid lineage. Carp SITR is abundantly expressed in macrophages and is secreted upon in vitro stimulation with the protozoan parasite T. borreli. Secretion of SITR protein during in vivo T. borreli infection suggests a role for this IgSF receptor in the host response to this protozoan parasite. Overexpression of carp SITR in mouse macrophages and knock-down of SITR protein expression in carp macrophages, using morpholino antisense technology, provided evidence for the involvement of carp SITR in the parasite-induced NO production. Conclusion/Significance - We report the structural and functional characterization of a novel soluble immune-type receptor (SITR) in a teleost fish and propose a role for carp SITR in the NO-mediated response to a protozoan parasite

Evolutionary conservation of alternative activation of macrophages: structural and functional characterization of arginase 1 and 2 in carp (Cyprinus carpio L.)

Classically activated macrophages (caMF) play an important role in type-I immune responses and alternatively activated macrophages (aaMF) function in type-II immune responses. While the classical activation of fish macrophages has been well described, the existence of aaMF has not yet been described for teleosts. Arginase is the characteristic enzyme in aaMF and two isoforms have been described for mammals. To study the presence of aaMF in a primitive vertebrate species we cloned arginase 1 and 2 cDNA of common carp. Carp arginase 1 is a 340 aa protein with 63% aa sequence identity to human arginase 1. Carp arginase 2 is a 347 aa protein with 63% aa sequence identity to human arginase 2. Three highly homologous arginase 2 genes were found, each showing only single non-synonymous substitutions. Basal arginase 1 expression is mainly found in carp mid kidney. In contrast, arginase 2 was expressed in all organs examined with the highest basal gene expression in liver. Cultured carp head kidney-derived macrophages were used to study aaMF in vitro. Carp macrophages showed significant arginase activity which could be induced by dibutyryl cyclic adenosine mono phosphate (cAMP) and specifically inhibited by NG-hydroxy-l-arginine (NOHA). At the gene level, arginase 2 gene expression was upregulated by cAMP stimulation, while arginase 1 gene expression was not influenced. LPS stimulation did not alter the arginase 1 or 2 expression, inducible nitric oxide synthase (iNOS) expression was, however, upregulated. This expression of iNOS was used as a measure of classical activation of carp macrophages. Thus, in contrast to mammals, fish arginase 2 and not arginase 1 is differentially regulated and likely involved in the alternative activation of fish macrophages. Our data suggest there may be an evolutionary conservation of the presence of aaMF down to teleost fish

Parasite infections revisited

Studying parasites helps reveal basic mechanisms in immunology. For long this has been recognized for studies on the immune system of mice and man. But it is not true for immunological studies on fish. To support this argument we discuss selected examples of parasite infections not only in warm-blooded but also in cold-blooded vertebrates. We point out that parasite infections deserve more attention as model systems in comparative immunolog

Biosynthesis of costunolide, dihydrocostunolide, and leucodin. Demonstration of cytochrome P450-catalyzed formation of the lactone ring present in sesquiterpene lactones of chicory

Chicory (Cichorium intybus) is known to contain guaianolides, eudesmanolides, and germacranolides. These sesquiterpene lactones are postulated to originate from a common germacranolide, namely (+)-costunolide. Whereas a pathway for the formation of ger