Prolactin-induced production of reactive oxygen species and IL-1ß in leukocytes from the bony fish gilthead seabream involves Jak/Stat and NF-κB signaling pathways.

Prolactin (PRL), a peptide hormone produced by the pituitary gland, was shown to play an important role in the modulation of the immune system of lower and higher vertebrates. To further investigate the effects of PRL on the activation of professional phagocytes of bony fish, we stimulated head kidney leukocytes and purified macrophages from the gilthead seabream (Sparus aurata L.) with various physiological concentrations of native salmon PRL for 2 and 16 h and analyzed the respiratory burst activity and proinflammatory cytokine expression profile. The results showed that PRL was able to induce the production of reactive oxygen species and the expression of IL-1ß and TNF-α in a similar way to two major pathogen-associated molecular patterns: polyinosinic-polycytidylic acid and genomic DNA from the bacterium Vibrio anguillarum. Interestingly, when the leukocytes were stimulated with suboptimal concentrations of PRL in the presence of bacterial DNA, the expression of IL-1ß was synergistically induced. More importantly, all PRL activities were blocked by neutralizing Abs to PRL, as well as by pharmacological inhibitors of the Jak/Stat and NF-κB signaling pathways. In addition, EMSA and HPLC/mass spectrometry further confirmed that Stat and NF-κB were involved in the activation of seabream leukocytes by PRL. Collectively, our data identified PRL as a key regulator of the activation of fish professional phagocytes and demonstrated a cross-talk between TLR/NF-κB and PRLR/Jak/Stat signaling pathways. In addition, to the best of our knowledge, this is the first report showing that PRL modulates the activation of phagocyte NADPH oxidase through the Jak/Stat pathway in vertebrates.

Prolactin peptide (pPRL) induces anti-prolactin antibodies, ROS and cortisol but suppresses specific immune responses in rainbow trout.

Prolactin has several immune functions in fish however, the effects on innate and specific components of rainbow trout immunity are currently unknown. Therefore in this study, prolactin peptide (pPRL) injection in rainbow trout generated anti-PRL antibodies that were confirmed through Western blot assays of fish brain tissue extract. At the same time, this group of fish was immunized with a viral antigen (VP2) and the specific antibody titer generated by the rainbow trout was subsequently determined, as well as the sero-neutralizing capacity of the antibodies. Interestingly, this group of fish (pPRL-VP2) generated approximately 150% less antibodies compared with fish immunized only with the viral antigen (VP2), and pPRL-VP2 fish increased their cortisol level by 4 times compared to the control. Additionally, through qPCR assay, we determined that the pPRL-VP2 fish group decreased pro-inflammatory transcript expression, and the serum of these (pPRL-VP2) fish stimulated ROS production in untreated fish leukocytes, a phenomenon that was blocked by the pharmacological cortisol receptor inhibitor (RU486). Collectively, this is the first report that indicates that pPRL could modulate both components of immunity in rainbow trout.

BVDV-1 induces interferon-beta gene expression through a pathway involving IRF1, IRF7, and NF-κB activation.

The bovine viral diarrhea virus (BVDV-1) is a pathogen with the capacity to modulate the interferon type I system. To further investigate the effects of BVDV-1 on the production of the immune response, the Madin-Darby bovine kidney cell line was infected with the cytopathic CH001 field isolate of BVDV-1, and the IFNbeta expression profiles were analyzed. The results showed that cpBVDV-1 was able to induce the production of IFNbeta in a way similar to polyinosinic-polycytidylic acid, but with less intensity. Interestingly, all cpBVDV-1 activities were blocked by pharmacological inhibitors of the IRF-1, IRF-7, and NF-κB signaling pathway, and the level of IFNbeta decreased at the level of transcript and protein. These results, together with in silico analyses showing the presence of several regulatory consensus target motifs, suggest that cpBVDV-1 regulates IFNbeta expression in bovines through the activation of several key transcription factors. Collectively, the results suggest that during cpBVDV-1 infection, cross talk is evident between various signaling pathways involved in transcriptional activation of IFNbeta in cattle.

Sumoylation of nucleoprotein (NP) mediated by activation of NADPH oxidase complex is a consequence of oxidative cellular stress during infection by Infectious salmon anemia (ISA) virus necessary to viral progeny.

The study evaluated the effects of nucleoprotein viral and the infectious virus in SHK-1 cells. The results show a strong respiratory burst activation and the induction of p47phox, SOD, GLURED, and apoptotic genes. Additionally, the cells alter the profile of SUMOylated proteins by the effect of transfection and infection experiments. In silico analyses show a set of structural motifs in NP susceptible of post-translational modification by the SUMO protein. Interestingly, the inhibition of the NADPH oxidase complex blocked the production of reactive oxygen species and the high level of cellular ROS due to the nucleoprotein and the ISAv. At the same time, the blocking of the p38MAPK signaling pathway and the use of Aristotelia chilensis, decreased viral progeny production. These results suggest that the NP triggers a strong production of ROS and modifying the post-translational profile mediated by SUMO-2/3, a phenomenon that favors the production of new virions.

Molecular characterization of the bovine IER3 gene: Down-regulation of IL-8 by blocking NF-κB activity mediated by IER3 overexpression in MDBK cells infected with bovine viral diarrhea virus-1.

The immediate early response 3 (IER3) is a key regulatory factor in the immune response, particularly as related to homeostasis immunomodulation via the nuclear factor kappa B (NF-κB) signaling pathway. The IER3 gene has been identified in mammals and, more recently, in other higher vertebrates. Nevertheless, relatively little is known about this regulator in bovines. Therefore, this study explored, characterized, and compared the genetic context of bovine IER3 to homologous genes in the human, mouse, and canine chromosomes. In silico analysis identified several regions of interest preserved in phylogenetically distant species. Similar analyses were also conducted for interleukin-8, a cytokine in which several putative cis elements were identified for the inducible transcription factor NF-κB. Subsequent challenge assays against the bovine viral diarrhea virus-1 revealed NF-κB signaling pathway activation just 15min post-infection, a process blocked by the BAY-117085 inhibitor. Similarly, infection strongly increased IER3 expression. Interestingly, IER3 down-regulated interleukin-8 expression, as confirmed by IER3 gene inhibition using small interfering RNA, RT-qPCR, and luciferase assays. In conclusion, this is the first report to present data indicating that bovine IER3 is a strong regulator of immune-marker expression, specifically modulating bovine interleukin-8 activation through the NF-κB/IER3 pathway in response to the bovine viral diarrhea virus.

Infectious pancreatic necrosis virus infection of fish cell lines: Preliminary analysis of gene expressions related to extracellular matrix remodeling and immunity.

The pathogenic infectious pancreatic necrosis virus (IPNV) causes high economic losses in fish farming. This virus can modulate several cellular processes during infection, but little is known about the infection mechanism. To investigate gene activation in response to IPNV, CHSE/F and SHK-1 cell line were infected with a cytopathic Sp field isolate of IPNV, and the expression profiles of proinflammatory, antiviral cytokine, and extracellular matrix markers were analyzed. IPNV induced the production of perlecan, fibulin-1, matrix metalloproteinase-2, 14-3-3ß, interleukin-1ß, Mx1, and interferon regulatory factors-1, -3, and -9. Interestingly, IPNV-mediated activity was blocked by pharmacological inhibitors of the NF-κB signaling pathway. These results, together with in silico analyses showing the presence of several regulatory consensus-target motifs, suggest that IPNV regulates gene expressions in fish through the activation of several key transcription factors. Collectively, these data indicate that IPNV is a viral regulator of expression for extracellular-matrix and immune markers, even during early infection. Finally, this is the first report in fish to find IPNV modulating the activation of interleukin-1ß production primarily through the NF-κB pathway.

Bovine A20 gene overexpression during bovine viral diarrhea virus-1 infection blocks NF-κB pathway in MDBK cells.

Viruses have developed cellular strategies to ensure progeny survival. One of the most interesting is immune camouflage, where the virus triggers a controlled-intensity immune response that prevents total destruction of the infected cell, thus "winning time" for the virus. This study explored the regulatory contexts of the bovine A20 gene during bovine viral diarrhea virus (BVDV)-1 infection, using IL-8 as an immune-response sentinel molecule. Assessments were conducted through RT-qPCR, Western blotting, gene silencing/overexpression, luciferase assays, and the use of pharmacological inhibitors, among other approaches. The results demonstrated that a) BVDV-1 increased A20 levels in Madin-Darby bovine kidney cells, b) increased A20 led to decreased IL-8 expression, and c) the virus affected the NF-κB signaling pathway. Collectively, these data identify bovine A20 as a strong regulator of immune marker expression. In conclusion, this is the first report on BVDV-1 modulating bovine IL-8 activation through the NF-κB/A20 pathway.

Characterization of bovine A20 gene: Expression mediated by NF-κB pathway in MDBK cells infected with bovine viral diarrhea virus-1.

Cytokine production for immunological process is tightly regulated at the transcriptional and posttranscriptional levels. The NF-κB signaling pathway maintains immune homeostasis in the cell through the participation of molecules such as A20 (TNFAIP3), which is a key regulatory factor in the immune response, hematopoietic differentiation, and immunomodulation. Although A20 has been identified in mammals, and despite recent efforts to identify A20 members in other higher vertebrates, relatively little is known about the composition of this regulator in other classes of vertebrates, particularly for bovines. In this study, the genetic context of bovine A20 was explored and compared against homologous genes in the human, mouse, chicken, dog, and zebrafish chromosomes. Through in silico analysis, several regions of interest were found conserved between even phylogenetically distant species. Additionally, a protein-deduced sequence of bovine A20 evidenced many conserved domains in humans and mice. Furthermore, all potential amino acid residues implicated in the active site of A20 were conserved. Finally, bovine A20 mRNA expression as mediated by the bovine viral diarrhea virus and poly (I:C) was evaluated. These analyses evidenced a strong fold increase in A20 expression following virus exposure, a phenomenon blocked by a pharmacological NF-κB inhibitor (BAY 117085). Interestingly, A20 mRNA had a half-life of only 32min, likely due to adenylate- and uridylate-rich elements in the 3'-untranslated region. Collectively, these data identify bovine A20 as a regulator of immune marker expression. Finally, this is the first report to find the bovine viral diarrhea virus modulating bovine A20 activation through the NF-κB pathway.

Resistance-nodulation-division efflux pump acrAB is modulated by florfenicol and contributes to drug resistance in the fish pathogen Piscirickettsia salmonis.

Piscirickettsia salmonis is a fastidious intracellular pathogen responsible for high mortality rates in farmed salmonids, with serious economic consequences for the Chilean aquaculture industry. Oxytetracycline and florfenicol are the most frequently used antibiotics against P. salmonis, but routine use could contribute to drug resistance. This study identified differentiated florfenicol susceptibilities in two P. salmonis strains, LF-89 and AUSTRAL-005. The less susceptible isolate, AUSTRAL-005, also showed a high ethidium bromide efflux rate, indicating a higher activity of general efflux pump genes than LF-89. The P. salmonis genome presented resistance nodulation division (RND) family members, a family containing typical multidrug resistance-related efflux pumps in Gram-negative bacteria. Additionally, efflux pump acrAB genes were overexpressed in AUSTRAL-005 following exposure to the tolerated maximal concentration of florfenicol, in contrast to LF-89. These results indicate that tolerated maximum concentrations of florfenicol can modulate RND gene expression and increase efflux pump activity. We propose that the acrAB efflux pump is essential for P. salmonis survival at critical florfenicol concentrations and for the generation of antibiotic-resistant bacterial strains.

ISAV infection promotes apoptosis of SHK-1 cells through a ROS/p38 MAPK/Bad signaling pathway.

The impact that the infectious salmon anemia virus (ISAV) has on the immune response of Salmo salar, from the perspective of activating/inactivating cellular processes, is currently unknown. Therefore, the present study evaluated this interaction and found that SHK-1 cells infected with ISAV resulted in respiratory burst activation and the induction of a strong pro-apoptotic imbalance through an increased expression of the Bad protein and decreased transcripts of Bcl-xl. Interestingly, the pharmacological inhibition of the p38 MAPK protein through SB203580 blocked the production of reactive oxygen species, the activity of caspase 3, and the formation of apoptotic nuclei in SHK-1 cells. Additionally, when the NADPH oxidase complex, a producer of superoxide anions, was blocked through apocynin, decreased apoptotic activity was observed in infected cells without significant modifications to viral amplification. These results, together with bioinformatics analysis performed for the Bad gene of fugu, suggest that the ISA virus triggers a strong production of oxygen radicals capable of activating transduction signaling pathways and mediating the expression and activation of pro-apoptotic proteins through the p38 MAPK pathway, all of which results in the apoptosis of ISAV infected cells.

Cytopathic BVDV-1 strain induces immune marker production in bovine cells through the NF-κB signaling pathway.

The bovine viral diarrhea virus (BVDV-1) is a pathogen responsible for high economic losses in the cattle industry worldwide. This virus has the capacity to modulate the immune system of several higher vertebrates, but there is little information available on the cell infection mechanism. To further investigate the effects of BVDV-1 on the activation of the immune response, the Madin-Darby bovine kidney cell line was infected with the cytopathic CH001 field isolate of BVDV-1, and the proinflammatory and antiviral cytokine expression profiles were analyzed. The results showed that BVDV-1 was able to induce the production of BCL3, IL-1ß, IL-8, IL-15, IL-18, Mx-1, IRF-1, and IRF-7 in a way similar to polyinosinic-polycytidylic acid. Interestingly, all BVDV-1 activities were blocked by pharmacological inhibitors of the NF-κB signaling pathway. These results, together with in silico analyses showing the presence of several regulatory consensus target motifs, suggest that BVDV-1 regulates gene expression in bovines through the activation of several key transcription factors. Collectively, these data identified BVDV-1 as a viral regulator of immune marker expression, even from early infection. Additionally, this is the first report to find BVDV-1 modulating the activation of cytokine production and transcriptions factors mainly through the NF-κB pathway in vertebrates.

The effects of reference genes in qRT-PCR assays for determining the immune response of bovine cells (MDBK) infected with the Bovine Viral Diarrhea Virus 1 (BVDV-1).

The bovine viral diarrhea virus (BVDV) causes significant economic losses to the dairy industry worldwide, and understanding its infection mechanisms would be extremely useful in designing new and efficient treatments. Due to the limited number of specific antibodies against bovine proteins, differential gene expression analyses are vital for researching host immune responses to viral infection. qRT-PCR provides a sensitive platform to conduct such gene expression analyses, but suitable housekeeping genes are needed for accurate transcript normalization. The present study assessed nine reference genes in bovine kidney cells under conditions of BVDV-1 infection, incubation with pathogen-associated molecular patterns, and co-incubation with BAY117085, a pharmacological inhibitor of the NF-κB signaling pathway. Analyses of Ct values using the BestKeeper and Normfinder programs ranked CD81, RPL4, and GAPDH as the most reliable reference genes. This determination of a stable set of reference genes in this culture system will facilitate analyses of expression levels for genes of interest.

ISA virus regulates the generation of reactive oxygen species and p47phox expression in a p38 MAPK-dependent manner in Salmo salar.

Several viruses, including Orthomyxovirus, utilize cellular reactive oxygen species (ROS) for viral genomic replication and survival within host cells. However, the role of ROS in early events of viral entry and signal induction has not been elucidated. Here, we show that ISA virus (ISAV) induces ROS production very early during infection of CHSE-214 and SHK-1Ycells, and that production is sustained over the observed 24h post-infection. The mitogen-activated protein kinase (MAPK) family is responsible for important signaling pathways. In this study, we report that ISAV activates ERK and p38 in Salmo salar. In salmonid macrophages, while ERK was required for SOD, GLURED, p47phox expression, p38 regulated the ROS production by the NADPH oxidase complex activation. These results, together with the presence of several consensus target motifs for p38 MAPK in the promoter of the S. salar p47phox gene, suggest that p38 MAPK regulates p47phox gene expression in fish through the activation of this key transcription factor.

Draft Genome Sequence of Virulent Strain AUSTRAL-005 of Piscirickettsia salmonis, the Etiological Agent of Piscirickettsiosis.

We report here the draft genome sequence of a lethal pathogen of farmed salmonids, Piscirickettsia salmonis strain AUSTRAL-005. This virulent strain was isolated in 2008 from Oncorhynchus mykiss farms, and multiple genes involved in pathogenicity, environmental adaptation, and metabolic pathways were identified.

Induction of genes encoding NADPH oxidase components and activation of IFN regulatory factor-1 by prolactin in fish macrophages.

The role played by prolactin (PRL) in fish immunity is scant. We report here that stimulation of the Atlantic salmon monocytic cell line SHK-1 with native salmon PRL resulted in activation of the respiratory burst and induction of the expression of the genes encoding the phagocyte NADPH oxidase components p47phox, p67phox and gp91phox, and the transcription factor IFN regulatory factor-1 (IRF-1). Interestingly, the pharmacologic inhibition of the Jak/Stat signaling pathway with AG490 blocked reactive oxygen species (ROS) production, and the induction of genes encoding the NADPH oxidase components and IRF-1 in PRL-activated SHK-1 cells. In addition, PRL promoted the phosphorylation of Stat and induced the DNA binding activity of IRF-1. These results, together with the presence of several consensus target motifs for Stat and IRF-1 in the promoter of the tilapia p47phox gene, suggest that PRL regulates p47phox gene expression in fish through the activation of these two key transcription factors. Taken together, our results demonstrate that PRL induces the expression of the genes encoding the major phagocyte NADPH oxidase components and ROS production in fish macrophages via the JAK2/Stat/IRF-1 signaling pathway.

Prolactin-induced activation of phagocyte NADPH oxidase in the teleost fish gilthead seabream involves the phosphorylation of p47phox by protein kinase C.

The pituitary hormone prolactin (PRL) is a multifunctional polypeptide which act as a key component of the neuroendocrine-immune loop and as a local regulator of the macrophage response. The involvement of PRL in regulating monocyte/macrophage functions is suggested by the presence of PRL receptors in these cells. Recently, we reported that physiological concentrations of native PRL were able to induce the expression of the pro-inflammatory cytokines IL-1ß and TNFα, and the production of reactive oxygen species (ROS) in head kidney leukocytes and macrophages from the teleost fish gilthead seabream (Sparus aurata L.). In this study, we show that the NADPH oxidase subunit p47phox becomes phosphorylated in leukocytes stimulated with PRL, an effect that is blocked when neutralizing polyclonal antibodies to PRL are added. Additionally, the pharmacological inhibition of either protein kinase C (PKC) with calphostin C or the Jak/Stat signaling pathway with AG490 impaired PKC activation, p47phox phosphorylation and ROS production in seabream leukocytes activated with PRL. Taken together, our results demonstrate for the first time the need for PKC in regulating the PRL-mediated phosphorylation of p47phox, the activation of NADPH oxidase and the production of ROS by macrophages in vertebrates.

Prolactin-releasing peptide is a potent mediator of the innate immune response in leukocytes from Salmo salar.

Prolactin (PRL)-releasing peptide (PrRP) is a strong candidate stimulator of pituitary PRL transcription and secretion in teleosts. However, the role in control of extrapituitary PRL expression or its effects on innate immunity are unclear even in mammals. To study the possible presence of PrRP in peripheral organs, PrRP expression patterns and their effect on innate immunity were characterised in SHK-1 cells and head kidney (HK) leukocytes purified from the salmonid, Salmo salar. We detected immunoreactive cells in leukocytes from blood and HK of S. salar and found that PrRP mRNA was abundantly expressed in these cells. We have recently reported that physiological concentrations of native PRL, downstream of neuropeptide PrRP were able to induce expression of pro-inflammatory cytokines and the production of reactive oxygen species (ROS) in HK leukocytes and macrophages from S. salar and Sparus aurata. It is of interest to note that in this work we have revealed that synthetic PrRP was able to induce expression of pro-inflammatory cytokines (interleukins) IL-1ß, IL-6, IL-8, IL-12 and PRL. We also show here that PrRP increased both (ROS) production and phagocytosis. Taken together, our results demonstrate for the first time that PrRP may be a local modulator of innate immune responses in leukocytes from S. salar.

Lipopolysaccharide primes the respiratory burst of Atlantic salmon SHK-1 cells through protein kinase C-mediated phosphorylation of p47phox.

The superoxide-producing NADPH oxidase complex of phagocytes plays a crucial role in host defenses against microbial infection. NADPH oxidase consists of a membrane heterodimeric protein, composed of gp91phox and p22phox, and the cytosolic proteins, p40phox, p47phox and p67phox. In the present study, we clone and sequence the full-length cDNAs coding for the Atlantic salmon (Salmo salar) phagocyte NADPH oxidase components, p47phox, p67phox and gp91phox, using a homology cloning approach. The sequences of these cDNAs showed that the S. salar p47phox, p67phox and gp91phox genes contained single open reading frames, which encoded predicted proteins of 413, 504 and 565 amino acids, respectively. Comparison of the deduced amino acid sequences showed that the S. salar p47phox, p67phox and gp91phox sequences shared 51, 45 and 68% identity with those of human components, respectively. Despite this relatively low homology between salmon and mammalian NADPH oxidase subunits, their functional domains are highly conserved. We also found that the mRNA levels of p47phox, p67phox and gp91phox expression were higher in immune-related tissues, such as kidney, spleen and gill. In addition, infection of the salmon macrophage cell line SHK-1 with Piscirickettsia salmonis induced the expression of p47phox, but had no effect on p67phox and gp91phox expression. Finally, we show for the first time in fish that activation of macrophages with lipopolysaccharide promotes the activation of protein kinase C, which in turn phosphorylates p47phox, leading to NADPH oxidase activation and reactive oxygen species generation. Collectively, these results suggest that the mechanisms of activation of phagocyte NADPH oxidase are well conserved from fish to mammals.