Head Kidney Cells Research Articles

Characterization of nuclear factor of activated T-cells-c3 (NFATc3) and gene expression of upstream-downstream signaling molecules in response to immunostimulants in Pacific red snapper cells

The nuclear factor of activated T cells (NFAT) proteins have crucial roles in the development and function of the immune system since they not only regulate activation of T cells but are also involved in the control of thymocyte development and T-cell differentiation. In this study, NFATc3 was characterized from the Pacific red snapper, Lutjanus peru. LpNFAtc3, which contains an open reading of 3300 bp frame coding for a protein of 1100 aa with a predicted molecular weight of 118.52 kDa. The predicted protein showed a conserved NFAT family structure with signature motifs and domains, sharing high identity (up to 76%) compared to other fish sequences. NFATc3 gene expression was analyzed by real time-PCR in head-kidney cells (leukocytes and lymphocytes) following yeast, zymosan and Vibrio parahaemolyticus stimulation along with the expression of upstream (ILF2, ILF3 and CaN) and downstream (CD3, TCRβ, IL-6 and IL-12) molecules. This study revealed a broad expression of NFATc3 with a relative strong expression in intestine and lymphocytes. The expression of NFATc3 was differentially up-regulated after stimulation with yeast in head-kidney leukocytes and after bacterial infection in lymphocytes at 24 h. Interestingly, the yeast and zymosan were able to activate ILF2, ILF3 and CaN mRNA gene expression in both kinds of cells. On the other hand, NFAT downstream genes such as CD3, TCRβ, IL-6 and IL-12 were significantly up-regulated in leukocytes stimulated with yeast or zymosan at 12 h; however in lymphocytes, this up-regulation was detected when cells reacted to V. parahaemolyticus stimuli at 24 h. Stimulating Pacific red snapper leukocytes with immunostimulants as yeast significantly up-regulated the expression of NFATc3, and up- and down-stream molecular genes and NFATc3 lymphocytes expression were potentially involved in responses to invasion of bacterial pathogens in an early immune response.

Functional activities of interferon gamma in large yellow croaker Larimichthys crocea

Interferon gamma (IFN-γ) is a T helper cell type 1 (Th1) cytokine that plays important roles in almost all phases of immune and inflammatory responses. Although IFN-γ gene in large yellow croaker Larimichthys crocea has been reported, little is known about its bioactivity. In this study, large yellow croaker IFN-γ (LycIFN-γ) gene was found to be constitutively expressed in all tissues tested, with the highest levels in blood and heart. Based on stimulation with polyinosinic-polycytidylic acid [poly (I:C)] or inactivated trivalent bacterial vaccine, LycIFN-γ mRNA was significantly increased in spleen and head kidney tissues. LycIFN-γ transcripts were also detected in head kidney granulocytes, primary head kidney macrophages (PKM), head kidney leukocytes, and large yellow croaker head kidney cell line (LYCK), and were significantly up-regulated by poly(I:C) or lipopolysaccharide (LPS) in head kidney leukocytes. Recombinant LycIFN-γ protein (rLycIFN-γ) produced in Escherichia coli could enhance respiratory burst responses in PKM. Furthermore, rLycIFN-γ not only induced the expression of iNOS gene and release of NO, but also up-regulated the expression of proinflammatory cytokines TNF-α and IL-1β in PKM. These findings therefore indicated that LycIFN-γ has a role in mediating inflammatory response. In addition, rLycIFN-γ could significantly up-regulate expression of IFN-γ receptor CRFB13, signal transduction factor STAT1, transcription factors IRF1 and T-bet, and Th1-related cytokines IFN-γ and IL-2 in head kidney leukocytes, suggesting that LycIFN-γ may have the potential to promote Th1 immune response in large yellow croaker. Taken together, our results show that LycIFN-γ may be involved in inflammatory response and promote Th1 immune response as its mammalian counterpart.

Draft Genome Sequence of Epilithonimonas sp. FP211-J200, Isolated from an Outbreak Episode on a Rainbow Trout (Oncorhynchus mykiss) Farm.

ABSTRACTHere, we report the draft genome sequence of Epilithonimonas sp. FP211-J200, isolated from rainbow trout head kidney cells. The size of the genome is 4,110,772 bp, with a G+C content of 37.1%. The Epilithonimonas sp. FP211-J200 genome has genes related to tetracycline and β-lactam resistance. This is the first reported Epilithonimonas species genome isolated from a fish host.

Temporal changes in innate immunity parameters, epinecidin gene expression, and mortality in orange-spotted grouper, Epinephelus coioides experimentally infected with a fish pathogen, Vibrio harveyi JML1

Changes in innate immunity parameters and epinecidin mRNA transcript levels were examined to characterize the non-specific immune response of E. coioides to pathogenic V. harveyi JML1 isolated from affected cage-cultured fish. After fish had been injected with bacteria at a dose causing 30% mortality, blood and tissue samples were collected at 0, 6, 12, 24, 48, 72, 96, 120, and 240 h post-infection (hpi) for assessment of indices such as the oxidative burst (OB) and phagocytic index (PI) of head kidney cells, and lysozyme activity (LYS) and total immunoglobulin (Total Ig) levels of the plasma. The epinecidin mRNA transcript levels (EGE) from skin, gills, liver, kidney, and spleen tissues were also determined by gel-based RT-PCR. Lastly, daily mortality (DM), liver total bacterial load (TBC), and presumptive Vibrio count (TVC) were monitored up to 240 hpi. The results revealed that bacteria proliferated rapidly in fish tissue, reaching peak densities at 24 hpi for both TBC and TVC but was on a downward trend thereafter. The pattern in fish mortality closely correlated with TBC and TVC. Total Ig, OB, and PI in E. coioides were suppressed in the early part of infection when V. harveyi load was high but recovered and later increased as bacterial density declined. LYS and EGE were consistently high and their activities were not hampered by bacterial infection. The study demonstrated that V. harveyi JML1 interacts with E. coioides by transiently inhibiting some immune parameters resulting in mortalities. However, consistently high LYS, upregulated EGE, and resurgent PI, OB and Total Ig conferred resistance and subsequent recovery in the fish. The study provides new insights on the interaction between E. coioides and V. harveyi JML1 that can aid in formulating health management strategies for groupers. Further studies on prophylactic interventions to enhance the innate immune response in grouper during infection with V. harveyi JML1 are suggested.

Black Scorpionfish (Scorpaena porcus) Hemopoiesis: Analysis by Flow Cytometry and Light Microscopy.

Cell suspensions of head kidney and spleen of black scorpionfish (Scorpaena porcus L.) have been studied using flow cytometry and light microscopy. On the basis of forward scatter (FS) and side scatter (SS) distribution and light microscopy, two main types of cells in the hemopoietic organs were identified: "small cells" (5.1-8.9 µm) and "large cells" (10.8-15.3 µm). Subpopulation of small cells was formed by thrombocytes, lymphocytes, and elements with functionally inactive nuclei. Euchromatin level in their nuclei was low and acidophilic cytoplasm corresponded to negligible nucleic acids content. No proliferative activity was observed using SYBR Green I fluorescence analysis. Morphological characteristics of these cells coincided with colony forming units of mammals. Large cells in head kidney consisted of two subpopulations of cells differing in granularity and DNA content. Proliferating blast cells and differentiating cells of all hemopoietic lines were identified among them. Macrophages and apoptotic cells were also detected in head kidney. In spleen large cells cluster mainly included aged red blood cells with extended lengthwise axis. Blast cells and differentiating elements in spleen were not observed. Anat Rec, 2017. © 2017 Wiley Periodicals, Inc. Anat Rec, 300:1993-1999, 2017. © 2017 Wiley Periodicals, Inc.

Phylogeny and expression analysis of C-reactive protein (CRP) and serum amyloid-P (SAP) like genes reveal two distinct groups in fish

The acute phase response (APR) is an early innate immune function that is initiated by inflammatory signals, leading to the release of acute phase proteins to the bloodstream to re-establish homeostasis following microbial infection. In this study we analysed the Atlantic salmon (Salmo salar) whole-genome database and identified five C-reactive protein (CRP)/serum amyloid P component (SAP) like molecules namely CRP/SAP-1a, CRP/SAP-1b, CRP/SAP-1c, CRP/SAP-2 and CRP/SAP-3. These CRP/SAP genes formed two distinct sub-families, a universal group (group I) present in all vertebrates and a fish/amphibian specific group (group II). Salmon CRP/SAP-1a, CRP/SAP-1b and CRP/SAP-1c and CRP/SAP-2 belong to the group I family whilst salmon CRP/SAP-3 is a member of group II. Gene expression analysis showed that the salmon CRP/SAP-1a as well as serum amyloid A-5 (SAA-5), one of the major acute phase proteins, were significantly up-regulated by recombinant cytokines (rIL-1β and rIFNγ) in primary head kidney cells whilst the other four CRP/SAPs remained refractory. Furthermore, SAA-5 was produced as the main acute phase protein (APP) in Atlantic salmon challenged with Aeromonas salmonicida (aroA(-) strain) whilst salmon CRP/SAPs remained unaltered. Overall, these data illustrate the potential different functions of expanded salmon CRP/SAPs to their mammalian homologues.

Zinc as a Signal to Stimulate Red Blood Cell Formation in Fish.

The common carp can tolerate extremely low oxygen levels. These fish store zinc in a specific zinc-binding protein presented in digestive tract tissues, and under low oxygen, the stored zinc is released and used as a signal to stimulate erythropoiesis (red blood cell formation). To determine whether the environmental supply of zinc to other fish species can serve as a signal to induce erythropoiesis as in the common carp, head kidney cells of four different fish species were cultured with supplemental ZnCl2. Zinc stimulated approximately a three-fold increase in immature red blood cells (RBCs) in one day. The stimulation of erythropoiesis by zinc was dose-dependent. ZnSO4 solution was injected into an experimental blood loss tilapia model. Blood analysis and microscopic observation of the blood cells indicated that, in vivo, the presence of additional zinc induced erythropoiesis in the bled tilapia. In the fish species studied, zinc could be used as a signal to stimulate erythropoiesis both in vitro and in vivo. The present report suggests a possible approach for the induction of red blood cell formation in animals through the supply of a certain level of zinc through either diet or injection.

In vitro modulation of Drimys winteri bark extract and the active compound polygodial on Salmo salar immune genes after exposure to Saprolegnia parasitica

The rapid development of the aquaculture industry has global concerns with health management and control strategies to prevent and/or treat diseases and increase sustainability standards. Saprolegniosis is a disease caused by Saprolegnia parasitica, and is characterized by promoting an immunosuppression in the host. This study evaluated in vitro the extract and one active compound (polygodial) of Drimys winteri, a Chilean medicinal tree as a potential early immunostimulatory aid in Saprolegniosis control. Atlantic salmon (Salmo salar) head kidney cells (ASK-1) were incubated with both extract and pure polygodial before exposure to S. parasitica mycelium, and the expression of the immune-related genes interleukin 1β (IL-1β), interferon α (IFNα), and major histocompatibility complex II (MHCII) was evaluated. Both evidenced immunomodulatory capacities by increasing gene expressions. This immunomodulation related to a mitigatory action counteracting the immunosuppressing effects of S. parasitica. Despite that most immune-related genes were up-regulated, the down-regulation of MHCII, characteristic of S. parasitica infection, was lessened by pre-incubation with the compounds. This study provides the first insight on the potential of D. winteri bark extract as a possible immunomodulatory and defensive strategy against this oomycete infection in fish.

Molecular and functional characterization of Toll-like receptor 21 in large yellow croaker (Larimichthys crocea)

Toll-like receptor 21 (TLR21) is a non-mammalian TLR that functions similar to mammalian TLR9 in recognizing CpG DNA. In the present study, we identified a TLR21 homologue, LycTLR21, from large yellow croaker (Larimichthys crocea). The complete coding sequence of LycTLR21 is 2946 nucleotides long, encoding a protein of 981 amino acids. The deduced LycTLR21 protein has typical TLR domain architecture, including a signal peptide, 13 leucine-rich repeats (LRRs) in the extracellular region, a transmembrane region, and a cytoplasmic Toll-Interleukin-1 receptor (TIR) domain. Phylogenetic analysis showed that LycTLR21 falls into a major clade formed by all fish TLR21 sequences and is closely related to TLR21 in Epinephelus coioides and Oplegnathus fasciatus. LycTLR21 mRNA was constitutively expressed in all tissues tested, with higher levels in immune-related tissues, such as spleen, head kidney, and gills. Upon stimulation with inactivated trivalent bacterial vaccine, LycTLR21 mRNA was significantly increased in these three tissues. Overexpression of a chimeric plasmid containing the extracellular domain of human cluster of differentiation 4 (CD4) and the transmembrane and cytoplasmic domains of LycTLR21 could activate NF-κB, but not IFN-β in Chinese hamster ovary (CHO) cells, suggesting that LycTLR21 could mediate activation of NF-κB. LycTLR21 could specifically recognize three CpG-oligodeoxynucleotides (CpG-ODNs), CpG-ODN 1826, 2006, and 2007, but not other CpG-ODNs detected, poly(I:C), lipopolysaccharide (LPS), and lipoteichoic acid (LTA-SA). These three CpG-ODNs were found to significantly up-regulate the expression of LycTLR21 and downstream proinflammatory cytokines IL-1β and IL-6 of NF-κB pathway in large yellow croaker head kidney (LYCK) cells. In addition, the expression levels of LycTLR21, c-Rel subunit of NF-κB, IL-1β and IL-6 genes were quickly increased in the spleen and head kidney by bacterial infection, suggesting that LycTLR21 signaling pathway may play a role in immune response to bacterial infection.

A novel role for pigment genes in the stress response in rainbow trout (Oncorhynchus mykiss).

In many vertebrate species visible melanin-based pigmentation patterns correlate with high stress- and disease-resistance, but proximate mechanisms for this trait association remain enigmatic. Here we show that a missense mutation in a classical pigmentation gene, melanocyte stimulating hormone receptor (MC1R), is strongly associated with distinct differences in steroidogenic melanocortin 2 receptor (MC2R) mRNA expression between high- (HR) and low-responsive (LR) rainbow trout (Oncorhynchus mykiss). We also show experimentally that cortisol implants increase the expression of agouti signaling protein (ASIP) mRNA in skin, likely explaining the association between HR-traits and reduced skin melanin patterning. Molecular dynamics simulations predict that melanocortin 2 receptor accessory protein (MRAP), needed for MC2R function, binds differently to the two MC1R variants. Considering that mRNA for MC2R and the MC1R variants are present in head kidney cells, we hypothesized that MC2R activity is modulated in part by different binding affinities of the MC1R variants for MRAP. Experiments in mammalian cells confirmed that trout MRAP interacts with the two trout MC1R variants and MC2R, but failed to detect regulation of MC2R signaling, possibly due to high constitutive MC1R activity.

Isolation and Characterization of Salmonid CD4+ T Cells.

This study reports the isolation and functional characterization of rainbow trout (Oncorhynchus mykiss) CD4-1(+) T cells and the establishment of an IL-15-dependent CD4-1(+) T cell line. By using Abs specific for CD4-1 and CD3ε it was possible to isolate the double-positive T cells in spleen and head kidney. The morphology and the presence of transcripts for T cell markers in the sorted CD4-1(+)CD3ε(+) cells were studied next. Cells were found to express TCRα, TCRβ, CD152 (CTLA-4), CD154 (CD40L), T-bet, GATA-3, and STAT-1. The sorted CD4-1(+) T cells also had a distinctive functional attribute of mammalian T lymphocytes, namely they could undergo Ag-specific proliferation, using OVA as a model Ag. The OVA-stimulated cells showed increased expression of several cytokines, including IFN-γ1, IL-4/13A, IL-15, IL-17D, IL-10, and TGF-β1, perhaps indicating that T cell proliferation led to differentiation into distinct effector phenotypes. Using IL-15 as a growth factor, we have selected a lymphoid cell line derived from rainbow trout head kidney cells. The morphology, cell surface expression of CD4-1, and the presence of transcripts of T cell cytokines and transcription factors indicated that this is a CD4-1(+) T cell line. To our knowledge, this is the first demonstration of the presence of CD4-1(+)CD3ε(+) T cells in salmonids. As in mammals, CD4-1(+) T cells may be the master regulators of immune responses in fish, and therefore these findings and the new model T cell line developed will contribute to a greater understanding of T cell function and immune responses in teleost fish.

Immunomodulation of Lactobacillus pentosus PL11 against Edwardsiella tarda infection in the head kidney cells of the Japanese eel (Anguilla japonica).

Wild and farm-raised fish can be simultaneously exposed to different types of pathogens in their habitats. Hence, it is important to study their effects, whether isolated or in combination. Therefore, the aim of this study was to evaluate the effects of Lactobacillus pentosus PL11 on the transcription of specific cytokine genes related to immune response, using Japanese eel macrophages as an invitro model. Head kidney leukocytes were isolated from Japanese eels and cell viability was determined using an MTT reagent. In addition, the Griess reagent was used to determine the nitric oxide (NO) production while, an enzyme-linked immunosobent assay (ELISA) and a quantitative polymerase chain reaction (qPCR) were utilized to quantify the level of proinflammatory cytokines. The results of the study indicated that infection by Edwardsiella tarda alone causes a higher rate of cell death and an increase in the production of proinflammatory cytokines, such as interleukin-1β (IL-1β, 822.67±29.48pgmL(-1)), interleukin-6 (IL-6, 13.57±0.55pgmL(-1)), and tumor necrosis factor-α (TNF-α, 2033.67±84.68pgmL(-1)). However, co-culture with L.pentosus PL11 downregulates the production of NO and the related IL-1β, IL-6, and TNF-α by 46%, 88.4%, 59%, and 77%, respectively. Quantification of the mRNA expression level revealed it to be consistent with the ELISA analysis. Hence, we infer that L.pentosus PL11 plays a significant role in the immunmodulation of the inflammatory responses that arise in fish owing to infection by pathogenic bacteria such as Edwardsiella tarda.

Hydrolyzed fish proteins modulates both inflammatory and antioxidant gene expression as well as protein expression in a co culture model of liver and head kidney cells isolated from Atlantic salmon (Salmo salar).

Hydrolyzed fish proteins (H-pro) contain high concentrations of free amino acids and low molecular peptides that potentially may benefit fish health. The following study aimed to test whether the water-soluble phase of H-pro could attenuate lipopolysaccharide (LPS) provoked inflammation in liver cells and head kidney cells isolated from Atlantic salmon. Cells were grown as mono cultures or co cultures to assess possible crosstalk between immune cells and metabolic cells during treatments. Cells were added media with or without H-pro for 2 days before LPS exposure and harvested 24h post LPS exposure. Respective cells without H-pro and LPS were used as controls. H-pro alone could affect expression of proteins directly as H-pro increased catalase protein expression in head kidney- and liver cells, regardless of culturing methods and LPS treatment. Leukotriene B4 (LTB4) production was also increased by H-pro in head kidney cells co cultured with liver cells. H-pro increased LPS induced interleukin 1β (IL-1β) transcription in liver cells co cultured with head kidney cells. All cultures of head kidney cells showed a significant increase in IL-1β transcription when treated with H-pro+LPS. H-pro decreased caspase-3 transcription in liver cells cultured co cultured with head kidney cells. Peroxisome proliferator activated receptor α (PPAR α) was upregulated, regardless of treatment, in liver cells co cultured with head kidney cells clearly showing that culturing method alone affected gene transcription. H-pro alone and together with LPS as an inflammation inducer, affect both antioxidant and inflammatory responses.

β-naphthoflavone interferes with cyp1c1, cox2 and IL-8 gene transcription and leukotriene B4 secretion in Atlantic cod (Gadus morhua) head kidney cells during inflammation.

The objective of this study was to evaluate how β-naphthoflavone interacts with lipopolysaccharide (LPS) and polyinosinic acid: polycytidylic acid (poly I: C) induced innate immune parameters as well as phase I and phase II detoxification enzymes in head kidney cells isolated from Atlantic cod. β-naphthoflavone is a pure agonist of aryl hydrocarbon receptor (AhR) while LPS and poly I: C are not. β-naphthoflavone was added to head kidney leukocytes alone or together with LPS or poly I: C and the responses were evaluated in terms of protein and gene expression. The results showed that β-naphthoflavone (25nM), with and without LPS, significantly induced cytochrome P450 (cyp1c) transcription in cod head kidney cells. β-naphthoflavone (100nM) in the presence of the virus mimic, poly I: C, also increased cyp1c1transcription. LPS induced cyp1c1, cyclooxygenase 2 (cox2), interleukin 1β (IL-1β), interleukin 6 (IL-6) and interleukin 8 (IL-8) transcription, genes that were not affected by the tested β-naphthoflavone concentrations alone. However, β-naphthoflavone (25 and 50nM) strengthened LPS induced cox2 and IL-8 transcription. Cod head kidney cells exposed to β-naphthoflavone concentrations ranging from 25 to 100nM, with and without LPS or poly I: C, expressed AhR protein. LPS or β-naphthoflavone (5-50nM) significantly induced leukotriene B4 (LTB4) secretion compared to control. In conclusion, this study suggests that β-naphthoflavone could interfere with LPS induced immune cell signaling in cod head kidney cells.

Immunostimulatory effects of prolactin on TLR1 and TLR5M in SHK-1 cells infected with Piscirickettsia salmonis.

The innate immune system is the first line of defense against infection by pathogens. It consists of various elements, including Toll-like receptors (TLRs), which recognize molecular patterns associated with pathogens and trigger the immune response, through activation of important transcription factors such as NF-κB, which are usually found sequestered in the cytoplasm by IκBα until it receives the release signal. Piscirickettsia salmonis causes piscirickettsiosis or salmonid rickettsial septicemia, a disease of great importance in Chile, representing 79.4% of the secondary mortality in important species such as Salmo salar, which is reflected in the Chilean economy. Prolactin (PRL) is a peptide hormone which has immunomodulating functions in mammals and some fish. Olavarría et al. (2010, J Immunol 185:3873-3883) determined its ability to increase the respiratory burst, its relationship with the JAK/STAT pathway, and the expression of interleukin IL-1β in Sparus aurata. Therefore, the present study was intended to establish a possible correlation and modulation between the signal transduction pathway of PRL (JAK/STAT), the pathways of NF-κB, and TLRs, in an infection caused by P. salmonis in salmon head kidney (SHK‑1) cells of S. salar. Stimulus with native PRL from S. salar was performed, and gene expression was analyzed for IL-1β, IκBα, TLR1, and TLR5M (membrane-bound form). In addition, the effect of PRL in the nuclear translocation of the transcription factor NF-κB and the possible involvement of JAK2 were analyzed by using a pharmacological inhibitor of this kinase. The results show a positive modulation of PRL in all analyzed genes and a significant increase in the translocation of NF-κB, recording a maximum at 2 h post-treatment, supporting the stimulatory hypothesis of PRL.

Cloning and expressional analysis of STAT1 and 3 genes in rock bream, Oplegnathus fasciatus

Rock bream (Oplegnathus fasciatus) aquaculture in Korea has been threatened by Rock bream iridovirus (RBIV), a megalocytivirus widely infected in various fish species. Major symptoms of RBIV infection is acute inflammation and enlargement of spleen. There is no available vaccine for RBIV though closely related red sea bream iridovirus has been successfully prevented by a commercial vaccine. In our previous study, proinflammatory cytokines and IFN1 gene expression was initially upregulated but later down-regulated by RBIV infection. This study was conducted to clone and analyse the expression of STAT (signal transducers and activators of transcription) 1 and 3. STATs are transcription factors involved in the regulation of immune genes including IFNs and the viral SOCS of megalocytivirus is known to play a key role to inhibit immune response by blocking STAT-JAK pathway. In phylogenetic analysis, rock bream STAT1 and 3 genes were highly homologous with those of mandarin fish, Siniperca chuatsi and yellow croaker, Larimichthys crocea. The expressional study of STAT1 and 3 genes was performed in head kidney upon RBIV infection and immune stimulants like LPS or polyI:C in vitro and in vivo. Head kidney cells were aseptically isolated from rock bream and treated with LPS, polyI:C, or different doses of RBIV and analyzed kinetics of gene expressions of rock bream. STAT1 gene expression was significantly upregulated by polyI:C at 4h and sustained at the high expression level until 24 h while upregulated by RBIV at 4 h but down-regulated at 24h. Meanwhile, STAT3 gene expression was not significantly affected by polyI:C though STAT3 gene expression was down-regulated at 24h by RBIV. In vivo study showed the down-regulation of STAT3 gene expression at day 4. Although viral SOCS is known to inhibit the STAT-JAK pathway at the protein level, the downregulation of STAT 1 and 3 gene expressions might be involved in the down-regulation of IFNI and Mx gene expression upon RBIV infection, resulting mass mortality of rock bream.

Expression kinetics of interferon stimulated genes in Atlantic salmon head kidney cells (ASK) nucleofected with Poly(I:C)

Viral diseases are of great concern in fish farming. Close to 20% of salmon put out to sea are lost during production and a large part of this is due to infections. Oil-adjuvented vaccines are available against some virus, but the efficiency under field conditions have been questioned. Our goal is to test known agonists for human Toll-like receptors (TLRs) in modified nanobeads carriers as adjuvants in different vaccines formulations, against fish viruses. The primary early response to viruses is mediated by interferons (IFNs) and interferon stimulated genes (ISGs). The interferon system is activated through the recognition of pathogen associated molecular patterns (PAMP) by conserved pattern recognition receptors (PRRs), such as TLRs. Poly (I:C) is a synthetic analog of double-stranded RNA (dsRNA) that mimics a viral infection in mammals and it is one of the most thoroughly studied PAMPs with respect to immunostimulatory potential in therapeutics or vaccines. Before developing a vaccine is necessary to understand more about how the immune system responds to these ligands. We therefore analyzed the expression of some interferon stimulated genes at different time points in Atlantic salmon head kidney cells (ASK) transfected by electroporation with Poly(I:C). Cells were transfected using Amaxa Nucleofector Kit with 5μg of Poly (I:C) or mock transfected with only buffer. After transfection, cells were plated and incubated for different time points (0, 6, 12, 24, 48, 72 hours) when samples of RNA were extracted for cDNA synthesis. Our results shows that there are large differences in the temporal pattern of IFN and ISG expression in salmon cells. QPCR analysis revealed strong upregulation of IFN alpha and TNF alpha during the first 6 hours after stimulation. Both Isg15 and Mx1 showed higher levels, comparing with control, in the first 12 hours and reached a peak at 24 hours after transfection. Mock transfections using only buffer without adding RNA or DNA also induced upregulation of some of the genes tested, especially TNF alpha. Endoplasmic reticulum (ER) stress induced by transfections was analyzed by a XBP-1 splicing assay analysis, but the results were not consistent. Lipofections and nucleofections are popular techniques for introducing RNA and DNA into cells to study both protein expression and immunity to cytoplasmic nucleic acids. Further studies are therefore necessary to quantify the magnitude and duration of cellular stress caused by these methods to separate their impact on effects mediated via PPRs .

Selective estrogen receptor modulators differentially alter the immune response of gilthead seabream juveniles.

17α-ethynylestradiol (EE2), a synthetic estrogen used in oral contraceptives and hormone replacement therapy, tamoxifen (Tmx), a selective estrogen-receptor modulator used in hormone replacement therapy, and G1, a G protein-coupled estrogen receptor (GPER) selective agonist, differentially increased the hepatic vitellogenin (vtg) gene expression and altered the immune response in adult gilthead seabream (Sparus aurata L.) males. However, no information exists on the effects of these compounds on the immune response of juveniles. This study aims, for the first time, to investigate the effects of the dietary intake of EE2, Tmx or G1 on the immune response of gilthead seabream juveniles and the capacity of the immune system of the specimens to recover its functionality after ceasing exposures (recovery period). The specimens were immunized with hemocyanin in the presence of aluminium adjuvant 1 (group A) or 120 (group B) days after the treatments ceased (dpt). The results indicate that EE2 and Tmx, but not G1, differentially promoted a transient alteration in hepatic vtg gene expression. Although all three compounds did not affect the production of reactive oxygen intermediates, they inhibited the induction of interleukin-1β (il1b) gene expression after priming. Interestingly, although Tmx increased the percentage of IgM-positive cells in both head kidney and spleen during the recovery period, the antibody response of vaccinated fish varied depending on the compound used and when the immunization was administered. Taken together, our results suggest that these compounds differentially alter the capacity of fish to respond to infection during ontogeny and, more interestingly, that the adaptive immune response remained altered to an extent that depends on the compound.

Effects of a peptide derived from the extreme carboxyl terminal of IL-8 on immune response genes in the SHK-1 cell line

Interleukin (IL)-8 is a chemokine principally characterized for being a leukocyte chemotactic. Recently, our laboratory chemically synthesized an alpha-helical peptide derived from the extreme carboxyl terminal of IL-8 that, due to physicochemical characteristics and functions, also evidences an antimicrobial effector function against Gram-negative bacteria such as Aeromona salmonicidina, Pseudomona aeruginosa, and Escherichia coli. In higher vertebrates, there is ample evidence for the role that antimicrobial peptides play in regulating the immune response. Therefore, the objective of our study was to determine if the peptide derived from IL-8 affects relevant immune genes. For this, the transcriptional expression of immune response genes were assessed in the Atlantic salmon (Salmo salar) head kidney (SHK-1) cell line when stimulated with the synthetic antimicrobial peptide derived from IL-8. Our results evidenced that the expression of IL-6 slightly increased, while the expressions of iNOS and IL-1β decreased. Moreover, this peptide did not appear to affect the expressional levels of IL-8 or TNF. Our results suggest that this peptide could directly influence the genetic expression of leukocytes, such as macrophages, through a downor upregulation of immune genes. In conclusion, the peptide derived from IL-8 would not only contribute towards limiting bacterial infection, but it would also have the ability to block macrophage activity and thereby likely participate in the end of the immune response. This studied was financed through Proyecto FONDECYT N° 3150678

Functional characterization of recombinant interleukin (IL)-17A/F1 in the Japanese pufferfish (Takifugu rubripes)

In mammals, interleukin (IL)-17A and IL-17F are hallmark inflammatory cytokines, which are expressed by Th17 cells. In contrast, teleost IL-17A and IL-17F homologs named as IL-17A/F has been identified, and its functional aspect is poorly understood. Here, the recombinant protein of the Japanese pufferfish, Fugu (Takifugu rubripes) IL-17A/F1 was produced and purified using mammalian cell line expression system where the protein folding was achieved correctly. The recombinant (r) IL-17A/F1 was added into head kidney (HK) and intestinal endothelial (IE) cells prepared from Fugu to stimulate the immune regulations, and then phagocytic activity, superoxide anion production by NBT assay, and cytokine gene expressions in the treated HK and IE cells at 0, 3, 6, 12, 24 and 48 hours post-stimulation (hps) were conducted. The rIL-17A/F1-treated both HK and IE cells showed an increased phagocytic activity at 24 hps. Superoxide anion production increased significantly at 48 hps in the HK cells and also increased moderately at 48 hps in the IE cells. Moreover, the rIL-17A/F1 displayed an ability to enhance expression of pro-inflammatory cytokine mRNAs, such as IL-1β, TNF-α and IL-6 in Fugu. The expression of IL-1β, TNF-α and IL-6 genes was significantly up-regulated during 3 to 12 hps. These bioactivity and gene expression results suggest that Fugu IL-17A/F1 probably plays an important role in promoting inflammatory and host innate immune responses as a pro-inflammatory cytokine in fish.