Iván Mulero

Evolution of the Inflammatory Response in Vertebrates: Fish TNF-α Is a Powerful Activator of Endothelial Cells but Hardly Activates Phagocytes

TNF-α is conserved in all vertebrate classes and has been identified in all taxonomic groups of teleost fish. However, its biological activities and its role in infection are largely unknown. Using two complementary fish models, gilthead seabream and zebrafish, we report here that the main proinflammatory effects of fish TNF-α are mediated through the activation of endothelial cells. Thus, TNF-α promotes the expression of E-selectin and different CC and CXC chemokines in endothelial cells, thus explaining the recruitment and activation of phagocytes observed in vivo in both species. We also found that TLR ligands, and to some extent TNF-α, were able to increase the expression of MHC class II and CD83 in endothelial cells, which might suggest a role for fish endothelial cells and TNF-α in Ag presentation. Lastly, we found that TNF-α increases the susceptibility of the zebrafish to viral (spring viremia of carp virus) and bacterial (Streptococcus iniae) infections. Although the powerful actions of fish TNF-α on endothelial cells suggest that it might facilitate pathogen dissemination, it was found that TNF-α increased antiviral genes and, more importantly, had little effect on the viral load in early infection. In addition, the stimulation of ZF4 cells with TNF-α resulted in increased viral replication. Together, these results indicate that fish TNF-α displays different sorts of bioactivity to their mammalian counterparts and point to the complexity of the evolution that has taken place in the regulation of innate immunity by cytokines.

Histamine is stored in mast cells of most evolutionarily advanced fish and regulates the fish inflammatory response

Mast cells are important as initiators and effectors of innate immunity and regulate the adaptive immune responses. They have been described in all classes of vertebrates and seem to be morphologically and functionally similar. However, early studies had shown that fish and amphibian mast cells were devoid of histamine. In this study, we take a fresh look at the evolution of histamine and find that the mast cells of fish belonging to the Perciformes order, the largest and most evolutionarily advanced order of teleosts, are armed with histamine. More importantly, histamine is biologically active in these fish where it is able to regulate the inflammatory response by acting on professional phagocytes. In addition, the actions of histamine in these immune cells seem to be mediated through the engagement of H1 and H2 receptors, which, together with the H3 receptor, are well conserved in bony fish. We propose that the storage of histamine in vertebrate mast cells and its use as an inflammatory messenger was established in primitive reptiles (Lepidosauria) ≈276 million years ago. This same feature seems to have developed independently in Perciform fish much more recently in the Lower Eocene, between 55 and 45 million years ago, a short period during which the great majority of Percomorph families appeared.

The antimicrobial peptides piscidins are stored in the granules of professional phagocytic granulocytes of fish and are delivered to the bacteria-containing phagosome upon phagocytosis

Antimicrobial peptides (AMPs) are increasingly recognized as a critical first line of defence against many pathogens. The genes encoding these peptides are expressed in numerous tissue and cell types from a wide variety of different species including mammals, amphibians, fish, and insects. In this study, we report that the AMPs called piscidins were primarily present in the mast cells (MCs) of fish and were only identified in fish belonging to the Order Perciformes. It is striking that histamine was seen to have a similar evolutionary history, since the only piscine MCs endowed with this molecule are in the Perciformes. We also show that both MCs and professional phagocytic granulocytes were armed with different piscidin molecules. In contrast, macrophages were devoid of these AMPs. More importantly, we found by immunoelectron microscopy that piscidins were delivered to the bacteria-containing phagosome of granulocytes upon phagocytosis, suggesting a role for these AMPs in the killing of both extracellular and intracellular pathogenic bacteria.

Detection of antimicrobial peptides related to piscidin 4 in important aquacultured fish

Epithelial surfaces of fish, such as the gut, skin and gills, comprise a large surface area for possible pathogen invasion. Antimicrobial peptides (AMPs), innate immunity components, play a significant role in protecting fish. Piscidins are a family of AMPs. In this study, we detected the presence of the recently discovered piscidin 4 via bug blot, Western blot, ELISA and/or immunohistochemistry in striped bass (Morone saxatilis), white bass (M. chrysops), European seabass (Dicentrarchus labrax), gilthead seabream (Sparus aurata), red drum (Sciaenops ocellatus), and barramundi (Lates calcarifer). Via bug blot, gill extracts from all species had antibacterial activity corresponding to the migration rate of piscidin 4. Western blotting showed that piscidin 4 immunoreactivity was greatest in striped bass gill extract. The concentrations of piscidin 4 detected by the ELISA in striped bass gill (approximately 20 microg/ml) were well within the levels that are inhibitory to important fish bacterial pathogens. Piscidin 4 was also detected via immunohistochemistry in all fish except barramundi. Piscidin 4-positive cells were identified as mast cells (MC), but not all MC were piscidin 4-positive. Species, age, size and physiological condition at sampling were some factors that might affect piscidin expression in different species. Our data provide strong evidence that piscidin 4 isoforms are present in all these commercially important species.

Characterization of macrophages from the bony fish gilthead seabream using an antibody against the macrophage colony-stimulating factor receptor.

Two major professional phagocyte populations have been described in fish, namely granulocytes and monocytes/macrophages. Although the distribution and localization of macrophages have been documented in several teleost species using mainly light and/or electron microscopy, the lack of appropriate markers for these cells has hampered our in-depth knowledge of their biology. We report here the generation of a monospecific rabbit polyclonal antibody against the gilthead seabream macrophage colony-stimulating factor receptor (Mcsfr), which is an excellent marker of macrophages in mammals and the zebrafish. The anti-Mcsfr has been found to be very useful in immunohistochemistry (IHC) to specifically immunostain the purified macrophages (adherent cells) obtained from the head-kidney as well as different cell populations in paraffin-embedded organs, including the head-kidney, spleen, thymus, gills and liver. Unexpectedly, however, no Mcsfr immunoreactive (Mcsfr(+)) cells were observed in the brain and intestine of the gilthead seabream. We also show that the distribution of Mcsfr(+) cells in the head-kidney and the spleen is unaltered following infection with the fish pathogenic bacterium Vibrio anguillarum and that the Il1b-producing cells in these two organs after infection are exclusively acidophilic granulocytes. Finally, as the epitope recognized by the anti-Mcsfr is well conserved, we illustrate the potential usefulness of this antibody in other teleost species, such as the European seabass.

Vaccination of larvae of the bony fish gilthead seabream reveals a lack of correlation between lymphocyte development and adaptive immunocompetence.

Fish eggs are released and embryos hatch into a pathogenically hostile environment, at a time when their immunological capacity is severely limited. Although the eggs are initially protected by the envelope as well as by several innate and adaptive immune substances, which are transferred to eggs during fish vitellogenesis, it seems that young specimens depend fundamentally on their innate defence mechanisms. Here we show in the gilthead seabream, an immunologically tractable teleost fish model, that the first lymphocyte marker genes, those coding for the two subunits for the recombination activating gene, were detected by RT-PCR around 21-27 days post-hatching (dph). In addition, the transcripts coding for the alpha and beta subunits of the T-cell receptor and the light and heavy chains of immunoglobulin M were detected at 27-48 dph. However, most innate immune genes analyzed were already expressed at hatching, including those coding for the toll-like receptors, pro- and anti-inflammatory molecules, antiviral and antibacterial factors, and phagocyte markers. Using the information from the gene expression study, we also examined the achievement of immunocompetence by analyzing the protection induced by a bacterin against the pathogenic bacterium Photobacterium damselae subsp. piscicida. The results show that vaccination of young larvae of this species by either immersion or oral routes resulted in increased susceptibility to infection of the specimens, and point to the lack of correlation between the achievement of immunocompetence and detection of the adaptive immunity markers.

Ultrastructure and function of stalks of the diatom Didymosphenia geminata

One of the most striking features of the diatom Didymosphenia geminata, which has increased markedly in abundance in a number of countries in recent years, is the very large branched stalks. In order to help understanding their role, an ultrastructural study was carried out on two populations, one from a stream in northern England and the other from a river on Vancouver Island, Canada. In both cases, the main part of the stalk had a central reticulate core surrounded by an outer region with dense fibres. A longitudinal structure in the uppermost part of the stalk just under the collar surrounding the base of the cell may perhaps correspond to a tube. The structure of the septa formed where branches divide is also described. Phosphomonoesterase activity known to be present in the stalks was shown to occur in the inner peripheral layers of the stalks and especially in the collar area. The results show that stalks have a complex structure suggesting their importance for their phosphatase activity to overcome low inorganic phosphate concentrations. Their large surface may function in herbivory avoidance, a better exposure of cells to turbulent conditions to increase nutrient uptake, adsorption of limiting elements and gas exchange.

Modulation of leukocytic populations of gilthead sea bream (Sparus aurata) by the intestinal parasite Enteromyxum leei (Myxozoa: Myxosporea)

SUMMARY The cellular mucosal and systemic effectors of gilthead sea bream (GSB) (Sparus aurata) involved in the acute immune response to the intestinal parasite Enteromyxum leei were studied in fish experimentally infected by the anal route. In the intestinal inflammatory infiltrates and in lymphohaematopoietic organs (head kidney and spleen) of parasitized fish, the number of plasma cells, B cells (IgM immunoreactive) and mast cells (histamine immunoreactive) were significantly higher, whereas the number of acidophilic granulocytes (G7 immunoreactive) decreased, compared with non-parasitized and unexposed fish. These differences were stronger at the posterior intestine, the main target of the parasite, and no differences were found in the thymus. In non-parasitized GSB, the percentage of splenic surface occupied by melanomacrophage centres was significantly higher. These results suggest that the cellular response of GSB to E. leei includes proliferation of leukocytes in lymphohaematopoietic organs and recruitment into intestines via blood circulation involving elements of innate and adaptive immunity. Acidophilic granulocytes and mast cells presented opposite patterns of response to the parasite infection, with an overall depletion of the former and an increased amount of the latter. Some differences between both cell types were also detected in regard to their granule density and cell morphology.

Immunolocalisation of microcystins in colonies of the cyanobacterium Rivularia in calcareous streams

The cyanobacterium Rivularia is often the dominant genus in unpolluted stretches of many calcareous streams. Previous studies have detected microcystins in field-collected colonies from Mediterranean streams in Spain. Because sheaths and mucilage represent a substantial part of the colonies, the localisation of microcystins within Rivularia colonies was tested with immunological methods to elucidate the role of mucilage in toxicity. Microcystins were localised inside the trichomes, in the filament sheaths and in the colonial mucilage. The presence of microcystins was also shown in some heterocysts, but no mircocystins were detected in multicellular hairs. We suggest that microcystins are important for a benthic organism growing slowly for much of the time, that some labour division may exists between the cells in the colony and that these immunological methods may be a useful alternative for microcystin detection.