Molecular mechanisms of hepcidin regulation in sea bass (Dicentrarchus labrax).

Hepcidin, an antimicrobial peptide described as a key regulator of iron metabolism, is known to respond in mammals to several stimuli, including iron overload, anemia, hypoxia and inflammation, through a number of molecular pathways. In order to understand the molecular pathways involved in the regulation of hepcidin expression in teleost fish, we have isolated for European sea bass (Dicentrarchus labrax) several coding sequences of known molecules involved on these pathways in mammals, namely jak3, stat3, tmprss6, bmp6, bmpr2, hjv, smad4, smad5, tfr1 and tfr2. The transcription levels of the isolated genes were evaluated by real-time PCR on fish subjected to experimental iron modulation (overload/deficiency) or infection with Photobacterium damsela. Results show that genes associated with the major pathway of the inflammatory response (IL6/JAK/STAT pathway) in mammals are also modulated in sea bass, being up-regulated during infection. Similarly, genes of the pathways classically associated with the response to variations in iron status (the HJV/BMP/SMAD and HFE/TfR pathways) are also modulated, mostly through down-regulation in iron deficiency and up-regulation during iron overload. Interestingly, many of these genes are also found to be up-regulated during infection, which may indicate a crosstalk between the known pathways of hepcidin regulation. These observations suggest the evolutionary conservation of the mechanisms of hepcidin regulation in teleost fish.

A model for acute iron overload in sea bass (Dicentrarchus labrax L.).

Evaluation of several parameters involved in iron metabolism was carried out after intraperitoneal (i.p.) injection with iron dextran (IDx) in sea bass (Dicentrarchus labrax L.). After treatment, a rapid mobilization of IDx from the peritoneal cavity to other organs was observed. This was followed by a modification of normal peripheral blood iron parameters. Total iron (TI) and transferrin saturation (TS) rose rapidly, to 4.14 microg/ml and 83.7%, respectively, on day 3. In contrast, unsaturated iron binding capacity (UIBC) dropped from 3.19 microg/ml (at day 0) to 0.90 microg/ml on day 3. Tissue iron content was determined by atomic absorption spectometry (AAS). Three days post-IDx injection, values of iron concentration in liver, spleen and head kidney were significantly higher than control values (15, 6 and 9-fold increase, respectively). Samples of liver, spleen and head kidney were processed for routine histology, and the Perl's method was used for iron staining. Histological sections of the IDx-treated animals showed iron deposition in all tissues studied. In the liver, the iron was evenly distributed over the whole organ, being present in the hepatocytes. In the head kidney and spleen, the iron deposition was mainly observed in the melanomacrophage centres (MMCs). The present study characterizes several parameters involved in iron metabolism, and develops a fish model, of iron overload, which can be used in further studies of iron toxicity and iron-induced susceptibility to bacterial infections.

Expression and temperature-dependent regulation of the beta2-microglobulin (Cyca-B2m) gene in a cold-blooded vertebrate, the common carp (Cyprinus carpio L.).

Expression of beta2-microglobulin (beta2m) in the common carp was studied using a polyclonal antibody raised against a recombinant protein obtained from eukaryotic expression of the Cyca-B2m gene. Beta2m is expressed on peripheral blood Ig+ and Ig lymphocytes, but not on erythrocytes and thrombocytes. In spleen and pronephros, dull- and bright-positive populations could be identified correlating with the presence of erythrocytes, thrombocytes, and mature leucocytes or immature and mature cells from the lympho-myeloid lineage, respectively. Thymocytes were shown to be comprised of a single bright-positive population. The Cyca-B2m polyclonal antiserum was used in conjunction with a similarly produced polyclonal antiserum to an MHC class I (Cyca-UA) alpha chain to investigate the expression of class I molecules on peripheral blood leucocytes (PBL) at different permissive temperatures. At 12 degrees C, a temporary downregulation of class I molecules was demonstrated, which recovered to normal levels within 3 days. However, at 6 degrees C, a lasting absence of class I cell-surface expression was observed, which could be restored slowly by transfer to 12 degrees C. The expression of immunoglobulin molecules on B cells was unaffected by temperature changes. The absence of the class I cell-surface expression was shown to be the result of a lack of sufficient Cyca-B2m gene transcription, although Cyca-UA mRNA was present at comparable levels at all temperatures. This suggests that class I expression is regulated by a temperature-sensitive transcription of the Cyca-B2m gene.

Expression of MhcCyca class I and class II molecules in the early life history of the common carp (Cyprinus carpio L.)

In this study transcription of class I alpha chain (Cyca-UA), beta2-microglobulin (Cyca-B2m) and class II alpha (Cyca-DXA) and beta (Cyca-DAB) during the early stages of embryo development was investigated by semiquantitative PCR. No transcripts of the genes under investigation were detected in the unfertilized egg. The expression of the genes encoding for the class II molecules revealed to be synchronized starting at day 1, unlike those for the class I molecules. Transcription of Cyca-B2m was first detected at day 7, whereas Cyca-UA was already present on day 1. This discrepancy would suggest absence of class I molecules during early development. The transcription of the Mhc genes in lymphoid organs was well established on day 21, with the exception of the spleen. In later stages of ontogeny cell surface expression of class I molecules was studied using polyclonal antibodies to Cyca-UA and Cyca-B2m in conjunction with detection of surface Ig. In week 3-10 Cyca-B2m was found on a higher percentage of cells from pronephros, spleen and thymus compared to Cyca-UA, suggesting the use of an alternative class I alpha chain. In the thymus, unlike the other organs, this difference remained present in the adult stage. The most likely candidates are alpha chains encoded by non-classical class I genes.

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.

Fish major histocompatibility complex genes: an expansion.

The advent of polymerase chain reaction technology has provoked a large amount of progress in the field of fish major histocompatibility complex (MHC) research. Many new teleost sequences have been reported in the last four years, including representatives of all classes of MHC genes. While the intron-exon structure of teleost MHC genes is now becoming clear, the organisation of the genes within the teleost MHC is still unclear. The sequences reported to date have been used for phylogenetic analysis and, due to their evolutionary position, are discussed in relation to hypotheses regarding the origin of the MHC. Teleost MHC gene sequences are also examined to see if conserved features of the both the nucleotide and amino acid sequences of higher vertebrate MHC genes are present. Differences in these features will reflect functional differences between teleost and mammalian MHC genes and may also have evolutionary implications.