Carolina Tafalla

Identification of a second group of type I IFNs in fish sheds light on IFN evolution in vertebrates.

In this report, three type I IFN genes were identified in rainbow trout (rt) Oncorhynchus mykiss and are classified into two groups based on their primary protein sequences: group I containing two cysteine residues; and group II containing four cysteines residues. The group I rtIFNs were induced in fibroblasts (RTG-2 cells), macrophages (RTS-11 cells), and head kidney leukocytes when stimulated with polyinosinic:polycytidylic acid, whereas group II IFN was up-regulated in head kidney leukocytes but not in RTG-2 and RTS-11 cells. Recombinant group I rtIFNs were potent at inducing Mx expression and eliciting antiviral responses, whereas recombinant group II rtIFN was poor in these activities. That two subgroups of type I IFN exist in trout prompted a survey of the genomes of several fish species, including zebrafish, medaka, threespine stickleback and fugu, the amphibian Xenopus tropicalis, the monotreme platypus and the marsupial opossum, to gain further insight into possible IFN evolution. Analysis of the sequences confirmed that the new IFN subgroup found in trout (group II IFN) exists in other fish species but was not universally present in fish. The IFN genes in amphibians were shown for the first time to contain introns and to conserve the four cysteine structure found in all type I IFNs except IFN-βε and fish group I IFN. The data overall support the concept that different vertebrate groups have independently expanded their IFN types, with deletion of different pairs of cysteines apparent in fish group I IFN and IFN-βε of mammals.

Chemokines in teleost fish species.

Chemokines are chemoattractant cytokines defined by the presence of four conserved cysteine residues which in mammals can be divided into four subfamilies depending on the arrangement of the first two conserved cysteines in their sequence: CXC (α), CC (β), C and CX(3)C classes. Evolutionarily, fish can be considered as an intermediate step between species which possess only innate immunity (invertebrates) and species with a fully developed acquired immune network such as mammals. Therefore, the functionality of their different immune cell types and molecules is sometimes also intermediate between innate and acquired responses. The first chemokine gene identified in a teleost was a rainbow trout (Oncorhynchus mykiss) chemokine designated as CK1 in 1998. Since then, many different chemokine genes have been identified in several fish species, but their role in homeostasis and immune response remains largely unknown. Extensive genomic duplication events and the fact that chemokines evolve more quickly than other immune genes, make it very difficult to establish true orthologues between fish and mammalian chemokines that would help us with the ascription of immune roles. In this review, we describe the current state of knowledge of chemokine biology in teleost fish, focusing mainly on which genes have been identified so far and highlighting the most important aspects of their expression regulation, due to the great lack of functional information available for them. As the number of chemokine genes begins to close down for some teleost species, there is an important need for functional assays that may elucidate the role of each of these molecules within the fish immune response.

Adjuvants and immunostimulants in fish vaccines: Current knowledge and future perspectives.

Vaccination is the most adequate method to control infectious diseases that threaten the aquaculture industry worldwide. Unfortunately, vaccines are usually not able to confer protection on their own; especially those vaccines based on recombinant antigens or inactivated pathogens. Therefore, the use of adjuvants or immunostimulants is often necessary to increase the vaccine efficacy. Traditional adjuvants such as mineral oils are routinely used in different commercial bacterial vaccines available for fish; however, important side effects may occur with this type of adjuvants. A search for alternative molecules or certain combinations of them as adjuvants is desirable in order to increase animal welfare without reducing protection levels. Especially, combinations that may target specific cell responses and thus a specific pathogen, with no or minor side effects, should be explored. Despite this, the oil adjuvants currently used are quite friendlier with respect to side effects compared with the oil adjuvants previously used. The great lack of fish antiviral vaccines also evidences the importance of identifying optimal combinations of a vaccination strategy with the use of a targeting adjuvant, especially for the promising fish antiviral DNA vaccines. In this review, we summarise previous studies performed with both traditional adjuvants as well as the most promising new generation adjuvants such as ligands for Toll receptors or different cytokines, focussing mostly on their protective efficacies, and also on what is known concerning their effects on the fish immune system when delivered in vivo.

Nitric oxide production by carpet shell clam (Ruditapes decussatus) hemocytes

We have demonstrated that carpet shell clam (Ruditapes decussatus) hemocytes produce nitric oxide (NO) in response to zymosan or bacterial lipopolysaccharide (LPS). This NO production was partially inhibited by the NO synthase inhibitor, N-omega-nitro-L-arginine (L-NAME). The capability of clam hemocytes to produce NO in response to the bacterial pathogen Vibrio tapetis was also studied. Incubation with bacteria induced a significant NO production by clam hemocytes, even though exogenous NO only slightly decreased the growth of V. tapetis. The effect of exogenous NO on the capability of clam hemocytes to phagocytose labeled Escherichia coli was studied using two different NO donors S-nitroso-N-acetyl-penicillamine (SNAP), and diethylenetriamine NO adduct (DETA/NO). Exogenous NO did not increase hemocyte phagocytosis, indicating that NO does not mediate phagocytosis in this species. These results are in accordance to those observed in other mollusk species, in which NO was independent of phagocytosis and constitutes an alternative method of killing invading pathogens.

Requirements for nitric oxide production by turbot (Scophthalmus maximus) head kidney macrophages

The effect of different cytokines and bacterial lipopolysaccharide (LPS) on turbot (Scophthalmus maximus) macrophage nitric oxide (NO) production has been studied. We have found two different responses concerning NO production in response to LPS. We have studied 43 turbot and only macrophage cultures derived from 30.2% of these turbot were significantly stimulated by LPS. The macrophage populations that did not respond to LPS, showed a constitutive production that was significantly reversed by NO inhibitors like N(G)-methyl-L-arginine (L-NMMA) and N-omega-nitro-L-arginine (L-NAME), and was dependent on intracellular calcium concentration. We studied the effect of other stimuli combined with LPS on the NO production of these otherwise non-responsive macrophages. LPS combined with turbot macrophage activating factor (MAF) containing supernatants, was capable of significantly stimulating some of these macrophage populations. The same response was observed when LPS was combined with turbot IFN-alphabeta-like substances. When LPS was combined with human recombinant tumor necrosis factor alpha (hrTNF-alpha), the NO production was significantly induced in all macrophage populations studied.

Production of nitric oxide by mussel (Mytilus galloprovincialis) hemocytes and effect of exogenous nitric oxide on phagocytic functions

In the present study, the ability of mussel (Mytilus galloprovincialis) hemocytes to produce nitric oxide (NO) in response to phorbol myristate acetate (PMA) was determined using the Griess reaction. Significant NO production was found in these cells in response to PMA. This stimulation was reversed in the presence of the NO synthase inhibitor, N(G)-methyl-L-arginine (L-NMMA). Moreover, the effect of the pre-incubation of hemocytes with NO was also determined on phagocytic immune functions of mussel hemocytes using two NO donors, glycerin trinitrate (GTN) and S-nitroso-N-acetyl-penicillamine (SNAP). In the case of GTN, a visible cytotoxic effect of the compound at the higher doses was observed. Those GTN concentrations that did not have a negative effect on hemocyte viability did not produce sufficient NO to significantly alter the chemiluminescent response to zymosan in all cases, nor the ability of hemocytes to phagocytose bacteria (Escherichia coli). SNAP, however, did not affect cell viability at either of the concentrations used and produced NO levels up to 13-fold higher than controls after 2 h of incubation. In this case, NO exogenously produced by SNAP significantly inhibited the chemiluminescent response of mussel hemocytes, whereas it did not have a significant effect on the capability of these cells to phagocytose bacteria.

Interleukin 8 and CK-6 chemokines specifically attract rainbow trout (Oncorhynchus mykiss) RTS11 monocyte–macrophage cells and have variable effects on their immune functions

In the current work, we have demonstrated that both rainbow trout (Oncorhynchus mykiss) interleukin 8 (IL-8), a CXC chemokine, and CK-6, a CC chemokine, are able of efficiently attracting RTS11, a rainbow trout established macrophage-monocyte-like cell line. Interestingly, two sub-populations of non-adherent cells are distinguishable by flow cytometry that could be identified as immature monocyte- and mature macrophage-like populations, respectively, and the two chemokines studied exert their effects on different populations. Although IL-8 specifically attracts the monocyte-like sub-population, CK-6 specifically attracts the macrophage-like cell sub-population. We have also determined the effects of both of these chemokines on RTS11 phagocytosis, respiratory burst and the expression of other immune-related genes. We found that IL-8 inhibited the phagocytosis capacity of RTS11 cells belonging to the macrophage-like profile. No effect was observed, however, on the respiratory burst, immune function that has been considerably affected throughout the establishment of the cell culture. Concerning the effect that IL-8 and CK-6 have on the expression of other immune genes, we found that IL-8 significantly induced the levels of expression of CK-6, IL-8, pro-inflammatory cytokines such as IL-1beta and tumour necrosis factor alpha (TNF-alpha) of RTS11 cells. On the other hand, CK-6 induced the levels of expression of IL-8, iNOS and the integrin CD-18, while it had very faint effect on pro-inflammatory cytokines. These results constitute one of the very few studies in which the effect of IL-8, a CXC chemokine, on monocyte-like cells is described. Moreover, it demonstrates that different monocyte-macrophage sub-populations have different reactivity to different chemokines.

Role of nitric oxide on the replication of viral haemorrhagic septicemia virus (VHSV), a fish rhabdovirus

In the present work, we have studied the role of nitric oxide (NO) on the replication of viral haemorrhagic septicemia virus (VHSV), a virus which produces high mortalities in fish aquaculture worldwide and that is known to replicate in turbot (Scophthalmus maximus) head kidney macrophages. Viral infection of turbot kidney macrophages in vitro induced an up-regulation of NO production and we have tested whether this endogenous NO production induced by VHSV on macrophages had an antiviral effect using the NO synthase inhibitor, N-omega-nitro-L-arginine (L-NAME). When L-NAME was added to the VHSV-infected cultures, no increase on VHSV titer was observed, even though the inhibitor was capable of decreasing NO production. When exogenous NO was apported by the nitric oxide donor, glycerin trinitrate (GTN) an antiviral effect on VHSV was observed. The NO donor significantly inhibited VHSV replication on a turbot fibroblast cell line (TV-1) and on turbot kidney macrophages.

The pyloric caeca area is a major site for IgM+ and IgT+ B cell recruitment in response to oral vaccination in rainbow trout

Although previous studies have characterized some aspects of the immune response of the teleost gut in response to diverse pathogens or stimuli, most studies have focused on the posterior segments exclusively. However, there are still many details of how teleost intestinal immunity is regulated that remain unsolved, including the location of IgM+ and IgT+ B cells along the digestive tract and their role during the course of a local stimulus. Thus, in the current work, we have studied the B cell response in five different segments of the rainbow trout (Oncorhynchus mykiss) digestive tract in both naïve fish and fish orally vaccinated with an alginate-encapsulated DNA vaccine against infectious pancreatic necrosis virus (IPNV). IgM+ and IgT+ cells were identified all along the tract with the exception of the stomach in naïve fish. While IgM+ cells were mostly located in the lamina propria (LP), IgT+ cells were primarily localized as intraepithelial lymphocytes (IELs). Scattered IgM+ IELs were only detected in the pyloric caeca. In response to oral vaccination, the pyloric caeca region was the area of the digestive tract in which a major recruitment of B cells was demonstrated through both real time PCR and immunohistochemistry, observing a significant increase in the number of both IgM+ and IgT+ IELs. Our findings demonstrate that both IgM+ and IgT+ respond to oral stimulation and challenge the paradigm that teleost IELs are exclusively T cells. Unexpectedly, we have also detected B cells in the fat tissue associated to the digestive tract that respond to vaccination, suggesting that these cells surrounded by adipocytes also play a role in mucosal defense.

CCR7 Is Mainly Expressed in Teleost Gills, Where It Defines an IgD+IgM− B Lymphocyte Subset

Chemokine receptor CCR7, the receptor for both CCL19 and CCL21 chemokines, regulates the recruitment and clustering of circulating leukocytes to secondary lymphoid tissues, such as lymph nodes and Peyers patches. Even though teleost fish do not have either of these secondary lymphoid structures, we have recently reported a homolog to CCR7 in rainbow trout (Oncorhynchus mykiss). In the present work, we have studied the distribution of leukocytes bearing extracellular CCR7 in naive adult tissues by flow cytometry, observing that among the different leukocyte populations, the highest numbers of cells with membrane (mem)CCR7 were recorded in the gill (7.5 ± 2% CCR7+ cells). In comparison, head kidney, spleen, thymus, intestine, and peripheral blood possessed <5% CCR7+ cells. When CCR7 was studied at early developmental stages, we detected a progressive increase in gene expression and protein CCR7 levels in the gills throughout development. Surprisingly, the majority of the CCR7+ cells in the gills were not myeloid cells and did not express membrane CD8, IgM, nor IgT, but expressed IgD on the cell surface. In fact, most IgD+ cells in the gills expressed CCR7. Intriguingly, the IgD+CCR7+ population did not coexpress memIgM. Finally, when trout were bath challenged with viral hemorrhagic septicemia virus, the number of CCR7+ cells significantly decreased in the gills while significantly increased in head kidney. These results provide evidence of the presence of a novel memIgD+memIgM− B lymphocyte subset in trout that expresses memCCR7 and responds to viral infections. Similarities with IgD+IgM− subsets in mammals are discussed.