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.

Consecutive emamectin benzoate and deltamethrin treatments affect the expressions and activities of detoxification enzymes in the rainbow trout (Oncorhynchus mykiss).

Rainbow trout (Oncorhynchus mykiss) subjected to three consecutive, alternating treatments with emamectin benzoate (EMB) and deltamethrin (DM) during outbreaks of Caligus rogercresseyi in a farm located in southern Chile (Hornopiren, Chiloé), were studied to determine the effects of these treatments on the protein and enzymatic activity levels of cytochrome P450 1A (CYP1A), flavin-containing monooxygenase (FMO) and glutathione S-transferase (GST) in different tissues. Consecutive and alternating EMB/DM treatments resulted in a 10-fold increase and 3-fold decrease of CYP1A protein levels in the intestine and gills, respectively. Notably, CYP1A activity levels decreased in most of the analyzed tissues. FMO protein and activity levels markedly increased in the kidney and the intestine. GST was up-regulated in all tissues, either as protein or enzyme activity. When comparing consecutive EMB/DM treatments against previous studies of EMB treatment alone, CYP1A activity levels were similarly diminished, except in muscle. Likewise, FMO activity levels were increased in most of the analyzed tissues, particularly in the muscle, kidney, and intestine. The increases observed for GST were essentially unchanged between consecutive EMB/DM and EMB only treatments. These results indicate that consecutive EMB/DM treatments in rainbow trout induce the expression and activity of FMO and GST enzymes and decrease CYP1A activity. These altered activities of detoxification enzymes could generate imbalances in metabolic processes, synthesis, degradation of hormones and complications associated with drug interactions. It is especially important when analyzing possible effects of consecutive antiparasitic treatments on withholding periods and salmon farming yields.

Comparative Pan-Genome Analysis of Piscirickettsia salmonis Reveals Genomic Divergences within Genogroups.

Piscirickettsia salmonis is the etiological agent of salmonid rickettsial septicemia, a disease that seriously affects the salmonid industry. Despite efforts to genomically characterize P. salmonis, functional information on the life cycle, pathogenesis mechanisms, diagnosis, treatment, and control of this fish pathogen remain lacking. To address this knowledge gap, the present study conducted an in silico pan-genome analysis of 19 P. salmonis strains from distinct geographic locations and genogroups. Results revealed an expected open pan-genome of 3,463 genes and a core-genome of 1,732 genes. Two marked genogroups were identified, as confirmed by phylogenetic and phylogenomic relationships to the LF-89 and EM-90 reference strains, as well as by assessments of genomic structures. Different structural configurations were found for the six identified copies of the ribosomal operon in the P. salmonis genome, indicating translocation throughout the genetic material. Chromosomal divergences in genomic localization and quantity of genetic cassettes were also found for the Dot/Icm type IVB secretion system. To determine divergences between core-genomes, additional pan-genome descriptions were compiled for the so-termed LF and EM genogroups. Open pan-genomes composed of 2,924 and 2,778 genes and core-genomes composed of 2,170 and 2,228 genes were respectively found for the LF and EM genogroups. The core-genomes were functionally annotated using the Gene Ontology, KEGG, and Virulence Factor databases, revealing the presence of several shared groups of genes related to basic function of intracellular survival and bacterial pathogenesis. Additionally, the specific pan-genomes for the LF and EM genogroups were defined, resulting in the identification of 148 and 273 exclusive proteins, respectively. Notably, specific virulence factors linked to adherence, colonization, invasion factors, and endotoxins were established. The obtained data suggest that these genes could be directly associated with inter-genogroup differences in pathogenesis and host-pathogen interactions, information that could be useful in designing novel strategies for diagnosing and controlling P. salmonis infection.

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.

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.

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.