Jana Montero

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.

Specific Regulation of the Chemokine Response to Viral Hemorrhagic Septicemia Virus at the Entry Site

ABSTRACT The fin bases constitute the main portal of rhabdovirus entry into rainbow trout (Oncorhynchus mykiss), and replication in this first site strongly conditions the outcome of the infection. In this context, we studied the chemokine response elicited in this area in response to viral hemorrhagic septicemia virus (VHSV), a rhabdovirus. Among all the rainbow trout chemokine genes studied, only the transcription levels of CK10 and CK12 were significantly upregulated in response to VHSV. As the virus had previously been shown to elicit a much stronger chemokine response in internal organs, we compared the effect of VHSV on the gills, another mucosal site which does not constitute the main site of viral entry or rhabdoviral replication. In this case, a significantly stronger chemokine response was triggered, with CK1, CK3, CK9, and CK11 being upregulated in response to VHSV and CK10 and CK12 being down-modulated by the virus. We then conducted further experiments to understand how these different chemokine responses of mucosal tissues could correlate with their capacity to support VHSV replication. No viral replication was detected in the gills, while at the fin bases, only the skin and the muscle were actively supporting viral replication. Within the skin, viral replication took place in the dermis, while viral replication was blocked within epidermal cells at some point before protein translation. The different susceptibilities of the different skin layers to VHSV correlated with the effect that VHSV has on their capacity to secrete chemotactic factors. Altogether, these results suggest a VHSV interference mechanism on the early chemokine response at its active replication sites within mucosal tissues, a possible key process that may facilitate viral entry.

Chemokine transcription in rainbow trout (Oncorhynchus mykiss) is differently modulated in response to viral hemorrhagic septicaemia virus (VHSV) or infectious pancreatic necrosis virus (IPNV)

Chemokines not only act as chemoattractants for immune cells, but also exert immunomodulatory actions, thus modulating the immune functions of their target cells. In rainbow trout (Oncorhynchus mykiss), twenty-four chemokines have been identified to date. Even though their sequences have been reported, their biological role has been scarcely elucidated and the role that these chemokines have on the antiviral response in fish has been poorly studied. In this sense, in the current work, we have determined the levels of expression of several of these rainbow trout chemokines (CXCd, gammaIP, CK1, CK3, CK5B, CK6, CK7A, CK9 and CK12) in head kidney and spleen during the course of a viral infection using two different viruses, viral hemorrhagic septicaemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV), comparing them to the levels induced by poly I:C. We also determined the effects that the two viruses and poly I:C provoked on the levels of expression of these chemokines in vitro in head kidney leucocytes. Overall, VHSV was capable of modulating gammaIP, CXCd, CK1, CK3, CK5B, CK6 and CK12, while IPNV induced a very different chemokine profile and affected CK1, CK5B, CK6, CK7A, CK9 and CK12. On the other hand, a viral mimic such as poly I:C was capable of up-regulating gammaIP, CXCd, CK1, CK3, CK5B, CK7A and CK12. As more information becomes available concerning the immune role and target cells that these chemokines have on rainbow trout, we would be able to better interpret the importance of these differences in the pathogenicity of these two viruses.

Viral hemorrhagic septicemia and infectious pancreatic necrosis viruses replicate differently in rainbow trout gonad and induce different chemokine transcription profiles.

Viral hemorrhagic septicemia virus (VHSV) and infectious pancreatic necrosis virus (IPNV) are two rainbow trout (Oncorhynchus mykiss) pathogens. While IPNV is known to be vertically transmitted to the next generation through the oocyte, VHSV is known to replicate in the ovary and be transmitted horizontally through the ovarian fluid. In this work, we wanted to study whether these differences had an effect on the immune response triggered in the ovary, with a focus on the chemokine response. We have studied the kinetics of viral gene expression and the sites of replication, confirming that great differences exist between the replication of the two viruses in the gonad. Next, we studied the levels of expression of several CXC and CC chemokines in the ovary and found that while VHSV strongly triggered chemokine transcription, IPNV had almost no effect. This lack of immune response might be an advantage that permits its vertical transmission.

CK12, a rainbow trout chemokine with lymphocyte chemo-attractant capacity associated to mucosal tissues

Although many chemokine genes have been identified in rainbow trout (Oncorhynchus mykiss) as in other teleost species, almost no studies focused on their biological role have been conducted, despite the fact that no clear inferences as to their functions can be made based on their low similarity to mammalian counterparts. In the current work, we have studied the regulation of mRNA transcription and protein expression of CK12, a rainbow trout CC chemokine previously catalogued within the CCL19/21/25 phylogenetic group. Our studies revealed that CK12 is strongly expressed both at mRNA and protein level in mucosal tissues. Mature lymphocyte populations also express CK12 both at mRNA and protein levels. Concerning its biological activity, a significant chemotatic activity towards purified recombinant CK12 in unfractionated leukocyte populations was observed in the spleen, but not in head kidney or blood. Consequently, a binding assay revealed that the number of leukocytes capable of binding CK12 was much more elevated in spleen populations than in leukocyte populations from other organs. This binding capacity was only observed in small lymphocytes that should account for resident inactivated lymphocytes, in contrast to mature lymphocytes that were responsible for CK12 production. Around 36% of these small lymphocytes were IgM+ cells, of which 40% had a CK12 binding capacity. On the other hand, 10% of thymocytes were also capable of CK12 binding, suggesting that both T and B immature lymphocytes are recruited by CK12. This work constitutes the first description of a mucosal-associated chemokine in fish in which important aspects of its regulation and functionality are revealed.

Immune effects observed after the injection of plasmids coding for rainbow trout (Oncorhynchus mykiss) CK5B, CK6 and CK7A chemokines demonstrate their immunomodulatory capacity and reveal CK6 as a major interferon inducer

In the current study, we have determined the immune effects of the intramuscular injection of eukaryotic expression plasmids coding for rainbow trout (Oncorhynchus mykiss) CK5B, CK6 or CK7A CC chemokines (pCK5B, pCK6 and pCK7A) as a first step towards the establishment of their biological role. We have studied the levels of expression of several immune genes in the spleen and head kidney by real-time PCR in comparison to the levels observed in animals injected with the empty plasmid. Concerning the levels of expression of these CC chemokines and the CXC chemokine, interleukin 8 (IL-8), each plasmid induced up-regulation on expression levels of its coded chemokine in the head kidney and spleen, but also affected the expression of other chemokines. Both pCK6 and pCK7A induced the expression of the other two CC chemokines, while pCK5B induced CK7A but not CK6. Both pCK5B and pCK7A induced IL-8 as well. pCK6 was the only plasmid that induced IL-1beta in the head kidney, whereas in the spleen, this occurred only with pCK5B. Different effects on the head kidney and spleen were also visible for tumour necrosis factor alpha (TNF-alpha), since the three plasmids induced this cytokine in the head kidney, but only pCK5B and pCK6 in the spleen. Concerning the effects on type I interferon (IFN), again pCK6 induced the strongest enhancement in the head kidney, while in the spleen it was pCK5B. However, the levels of expression of the Mx gene, know to be induced by type I IFN correlated with the CK6-induced IFN levels in the head kidney, but not with the CK5B-induced IFN in head kidney or spleen, suggesting an inhibition of Mx mRNA levels independent of IFN due to CK5B. The clear effect of pCK6 on the levels of expression of IFN-gamma and its strong effects on type I IFN, in contrast with its recent adscription to the CCL17/22 group linked to Th2 responses, were verified by studying the in vitro effects of recombinant CK6 on head kidney leukocytes. Again in this case, recombinant CK6 strongly induced type I IFN, Mx and IFN-gamma to a lesser extent, revealing CK6 as a potent IFN inducer in contrast to its mammalian homologues. Finally, effects on major histocompatibility complex (MHC)-IIalpha, CD4 and CD8alpha expression demonstrate that the three chemokines are able to mobilize antigen-presenting cells, CD4(+) and CD8(+) lymphocytes.

Prostaglandin E2 promotes M2 polarization of macrophages via a cAMP/CREB signaling pathway and deactivates granulocytes in teleost fish

The profile of prostaglandin (PG) production is determined by the differential expression of the enzymes involved in their production and degradation. Although the production of PGE2 by fish leukocytes has been relatively well studied in several fish species, knowledge of how its production is regulated, its biological activities and the signaling pathways activated by this PG is scant or even contradictory. In this work we show that in the teleost fish gilthead seabream (Sparus aurata L.) macrophages regulate PGE2 release mainly by inducing the expression of the genes encoding the enzymes responsible for its synthesis, while acidophilic granulocytes (AGs) not only induce these genes quickly after activation but also inhibit the expression of the genes encoding the enzymes responsible for PGE2 degradation at later time points. In addition, treatment of macrophages with PGE2 promoted their M2 polarization, which is characterized by high expression levels of interleukin-10, mannose-receptor c-type 1 and arginase 2 genes. In sharp contrast, PGE2 promoted the deactivation of AGs, since it decreased the production of reactive oxygen species and the expression of genes encoding pro-inflammatory cytokines. These differences are the result of the alternative signaling pathways used by PGE2 in macrophages and AGs, a cAMP/CREB signaling pathway operating in macrophages, but not in AGs, downstream of PGE2. Our data identify for the first time a role for professional phagocyte-derived-PGE2 in the resolution of inflammation in fish and highlight key differences in the PGE2 signaling pathway in macrophages and granulocytes.

Effects of recombinant flagellin B and its ND1 domain from Vibrio anguillarum on macrophages from gilthead seabream (Sparus aurata L.) and rainbow trout (Oncorhynchus mykiss, W.)

Flagellin is the principal component of flagellum in Gram negative and positive bacteria, and it is also the ligand that activates the Toll-like receptor 5 (TLR5) in mammals and fish. In higher vertebrates, flagellin induces the activation of the membrane-bound TLR5 (TLR5M), which promotes the expression of proinflammatory cytokines and chemokines and the co-stimulatory molecules present in antigen-presenting cells needed for the activation of T cells. In the present study, we report the production of two recombinant proteins of Vibrio anguillarum: i) a full length flagellin B (FlaB) (rFla) and ii) the amino-terminus of the D1 domain (rND1) of the same protein, the region mainly responsible for binding to TLR5 and for the immunostimulatory activity of flagellin. The effects of these recombinant proteins were assessed inxa0vitro using head kidney macrophages of gilthead seabream (Sparus aurata L., Perciformes, Sparidae) and rainbow trout (Oncorhynchus mykiss W., Salmoniformes, Salmonidae). In both species, 3xa0hxa0of stimulation with rFla and rND1 induced expression of the proinflammatory cytokines interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), and of the chemokine IL-8. In gilthead seabream macrophages stimulated with rFla and rND1, a 900- and 6-fold increase were observed for IL-1β transcription, while a 900- and 3-fold increase were recorded for IL-8 transcription, respectively, as compared to non-stimulated macrophages. In rainbow trout, rFla increased expression of IL-8 40-fold in macrophages, whereas rND1 increased expression of the chemokine 3-fold, as compared to non-stimulated cells. The results obtained for rFla and rND1 demonstrate their modulatory capabilities inxa0vitro, suggesting that rFla and rND1 could be evaluated as immunostimulatory candidates for use in farmed fish. However, further inxa0vivo studies are needed to confirm and expand on the present results.

Regulation of rainbow trout (Oncorhynchus mykiss) interleukin-8 receptor (IL-8R) gene transcription in response to viral hemorrhagic septicemia virus (VHSV), DNA vaccination and chemokines.

An interleukin-8 receptor (IL-8R)-like sequence has been previously reported in rainbow trout (Oncorhynchus mykiss); however, no further studies to confirm its biological activity or regulation of expression have been performed. In this report, we have studied the regulation of the transcription of this receptor in response to different stimuli both in vivo and in vitro. We found that in response to a viral hemorrhagic septicemia virus (VHSV) infection, the levels of expression of IL-8R are suppressed in the head kidney, spleen and muscle, in contrast to what occurs in response to Poly I:C. These results might indicate a suppressive effect of the virus and a mechanism that enables it to elude the immune response. This response is no longer observed in vitro in the rainbow trout macrophage cell line RTS11, which has been shown to be resistant to VHSV complete replication, and where the virus produced no effect on the levels of mRNA expression of IL-8R. In these cells, as observed in vivo, Poly I:C significantly induced the expression of IL-8R, increase that came along with an increase in the chemotactic activity towards IL-8. In response to DNA vaccination, we found that the levels of mRNA expression are significantly increased only in the muscle at very early times post-vaccination. As an additional step to clarify whether this receptor is in fact being used by IL-8, we intramuscularly injected plasmids coding for different rainbow trout chemokines (IL-8 and other CC chemokines such as CK5B, CK6 and CK7A). Only plasmids coding for IL-8 and CK-6 were capable of significantly increasing the levels of transcription of IL-8R in the muscle. This effect was confirmed by the up-regulation of IL-8R mRNA production in head kidney leucocytes in response to recombinant IL-8 and CK-6.

Professional phagocytic granulocyte-derived PGD2 regulates the resolution of inflammation in fish

Prostaglandins (PGs) play a key role in the development on the immune response through the regulation of both pro- and anti-inflammatory processes. PGD(2) can be either pro- or anti-inflammatory depending on the inflammatory milieu. Prostaglandin D synthase (PGDS) is the enzyme responsible for the conversion of PGH(2) to PGD(2). In mammals, two types of PGDS synthase have been described, the hematopoietic (H-PGDS) and the lipocalin (L-PGDS). In the present study we describe the existence of two orthologs of the mammalian L-PGDS (PGDS1 and PGDS2) in the gilthead seabream and characterize their gene expression profiles and biological activity. The results showed a dramatic induction of the gene coding for PGDS1 in acidophilic granulocytes (AGs), which are functionally equivalent to mammalian neutrophils, after a prolonged in vitro activation with different pathogen associated molecular patterns (PAMPs). In contrast PGDS2 was not expressed in these cells. The functional relevance of the induction of PGDS1 in AGs was confirmed by the ability of these cells to release PGD(2) upon PAMP stimulation. To gain further insight into the role of PGD(2) in the resolution of inflammation in fish, we examined the ability of PGD(2) or its cyclopentenone derivates (cyPGs) to modulate the main functional activities of AGs. It was found that both PGD(2) and cyPGs inhibited the production of reactive oxygen species and downregulated the transcript levels of the gene encoding interleukin-1β. Taken together, these results demonstrate that the use of PGD(2) and its metabolites in the resolution of inflammation was established before the divergence of fish from tetrapods more than 450 million years ago and support a critical role for granulocytes in the resolution of inflammation in vertebrates.