The insertion/deletion in the DNA-binding region allows the discrimination and subsequent identification of the glucocorticoid receptor 1 (gr1) and gr2 nucleotide sequences in gilthead sea bream (Sparus aurata): Standardizing the gr nomenclature for a better understanding of the stress response in teleost fish species

Cortisol carries out its physiological mechanism of action through the recognition by the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR) 1 (GR1) and GR2. Previous studies reported that the main difference between gr1 and gr2 nucleotide sequences resides in a 27-nucleotide insertion/deletion in the DNA-binding region, respectively. However, in gilthead sea bream (Sparus aurata) the annotation for gr1 and gr2 seems contradictory. The gr2 sequence possesses the characteristic 27-nucleotide insertion that, in fact, is associated with the gr1 nucleotide sequence. Thus, this study aimed to elucidate the nucleotide sequences for the gr1 and gr2 in gilthead sea bream. The Clustal Omega alignment for different fish species corroborated the presence of such 27-nucleotide insertion/deletion in the DNA-binding region for gr1 and gr2, respectively. Then, we design specific primers set for the amplification of the gilthead sea bream gr1 by polymerase chain reaction (PCR). Importantly, the gr1 nucleotide partial sequence has a high similarity with other gr1 sequences already published for other fish species, being present in all of them the 27-nucleotide insertion in the DNA-binding region. We also detected that in European sea bass the gr1 and gr2 sequences had not been named according to the 27-nucleotide insertion/deletion criteria in the DNA-binding region. Thus, our study makes an urgent call to the scientific community to discuss the establishment of an updated agreement that allows homogenizing the criteria for the nomenclature defining the gr1 and gr2 nucleotide sequences for a better understanding of the stress response in teleost fish species.This study thanks to the AGL2016-76069-C2-2- R, PID2020-117557RB-C21, PID2020-117557RB-C22 grants (AEI-MINECO; Spain). EV-V thanks the support of Fondecyt iniciacion grant (project number 11221308; Agencia Nacional de Investigacion y Desarrollo de Chile, Government of Chile). AK was the recipient of a Ministry of Science, Research, and Technology (Iran) fellowship. MT thanks for the support of the post-doctoral fellowship "Ramon y Cajal" (ref. RYC2019-026841-I) (Ministerio de Ciencia e Innovacion, Spanish Government). FER-L thanks the support of Fondecyt regular grant (project number: 1211841; Agencia Nacional de Investigacion y Desarrollo de Chile, Government of Chile)

The gene expression profile of the glucocorticoid receptor 1 (gr1) but not gr2 is modulated in mucosal tissues of gilthead sea bream (Sparus aurata) exposed to acute air-exposure stress

The perception of an acute stressor (short-duration; high-intensity) induces a physiological response that activates the hypothalamic-pituitary-interrenal (HPI) axis and the subsequent release of cortisol. Cortisol carries out its effect at the molecular level through its recognition by the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). Recently, we unveiled the nucleotide sequence of the glucocorticoid receptor 1 (gr1) and gr2 in gilthead sea bream (Sparus aurata). Importantly, GR1 and GR2 respond to different levels of cortisol concentration in fish and, consequently, play a differential role in the stress response. To date, and despite their relevance, no data describes the modulation of these receptors in response to an acute stressor in gilthead sea bream (S. aurata). In this study, we evaluated the kinetics of modulation of cortisol receptors expression (gr1, gr2, mr), and its similarity with the expression pattern of selected genes associated with stress (hsp70; enolase) and immune response (lysozyme; c3; il-1 beta; tnf-alpha; il-10; tgf-beta 1) in gilthead sea bream mucosal tissues (skin; gills; anterior gut). To do it, fish were acutely stressed by three-minute air exposure, and the expression profile was evaluated at zero, 1 h, 6 h, and 24 h post-stress (hps). The cortisol level in plasma and skin mucus peaked at 1 hps. All the mucosal tissues showed a time-dependent and tissue-specific upregulation of gr1 and mr. The immune-related genes showed the upregulation of il-1 beta at 6 hps (gills; anterior gut), and tnf-alpha and c3 at 24 hps (anterior gut). Taking together, our study concludes that fish subjected to three-minute air exposure modulated the expression of gr1 but not gr2 in mucosal tissues (skin; gills; anterior gut). Furthermore, our data reinforce the idea of a stimulatory effect induced in genes associated with the innate immune response after acute stress but focused at the mucosal level and in a time- and tissue-dependent manner

Non-Specific Antibodies Induce Lysosomal Activation in Atlantic Salmon Macrophages Infected by Piscirickettsia salmonis

Piscirickettsia salmonis, an aggressive intracellular pathogen, is the etiological agent of salmonid rickettsial septicemia (SRS). This is a chronic multisystemic disease that generates high mortalities and large losses in Chilean salmon farming, threatening the sustainability of the salmon industry. Previous reports suggest that P. salmonis is able to survive and replicate in salmonid macrophages, inducing an anti-inflammatory environment and a limited lysosomal response that may be associated with host immune evasion mechanisms favoring bacterial survival. Current control and prophylaxis strategies against P. salmonis (based on the use of antibiotics and vaccines) have not had the expected success against infection. This makes it urgent to unravel the host-pathogen interaction to develop more effective therapeutic strategies. In this study, we evaluated the effect of treatment with IgM-beads on lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The impact of IgM-beads on cytotoxicity induced by P. salmonis in infected cells was evaluated by quantification of cell lysis through release of Lactate Dehydrogenase (LDH) activity. Bacterial load was determined by quantification of 16S rDNA copy number by qPCR, and counting of colony-forming units (CFU) present in the extracellular and intracellular environment. Our results suggest that stimulation with antibodies promotes lysosomal activity by lowering lysosomal pH and increasing the proteolytic activity within this organelle. Additionally, incubation with IgM-beads elicits a decrease in bacterial-induced cytotoxicity in infected Atlantic salmon macrophages and reduces the bacterial load. Overall, our results suggest that stimulation of cells infected by P. salmonis with IgM-beads reverses the modulation of the lysosomal activity induced by bacterial infection, promoting macrophage survival and bacterial elimination. This work represents a new important evidence to understand the bacterial evasion mechanisms established by P. salmonis and contribute to the development of new effective therapeutic strategies against SRS

Non-lysosomal Activation in Macrophages of Atlantic Salmon (Salmo salar) After Infection With Piscirickettsia salmonis

Piscirickettsia salmonis is a facultative intracellular pathogen and etiological agent of the systemic disease salmonid rickettsial septicemia. It has been suggested that P. salmonis is able to survive in host macrophages, localized within a vacuole like-compartment which prevents lysosomal degradation. However, the relevant aspects of the pathogenesis of P. salmonis as the host modulation that allow its intracellular survival have been poorly characterized. In this study, we evaluated the role of lysosomes in the response to P. salmonis infection in macrophage-enriched cell cultures established from Atlantic salmon head kidneys. Bacterial infection was confirmed using confocal microscopy. A gentamicin protection assay was performed to recover intracellular bacteria and the 16S rDNA copy number was quantified through quantitative polymerase chain reaction in order to determine the replication of P. salmonis within macrophages. Lysosomal activity in Atlantic salmon macrophage-enriched cell cultures infected with P. salmonis was evaluated by analyzing the lysosomal pH and proteolytic ability through confocal microscopy. The results showed that P. salmonis can survive ≥120 h in Atlantic salmon macrophage-enriched cell cultures, accompanied by an increase in the detection of the 16S rDNA copy number/cell. The latter finding suggests that P. salmonis also replicates in Atlantic salmon macrophage-enriched cell cultures. Moreover, this bacterial survival and replication appears to be favored by a perturbation of the lysosomal degradation system. We observed a modulation in the total number of lysosomes and lysosomal acidification following infection with P. salmonis. Collectively, the results of this study showed that infection of Atlantic salmon macrophages with P. salmonis induced limited lysosomal response which may be associated with host immune evasion mechanisms of P. salmonis that have not been previously reported

Spray-Dried Porcine Plasma Promotes the Association Between Metabolic and Immunological Processes at Transcriptional Level in Gilthead Sea Bream (Sparus aurata) Gut

The spray-dried porcine plasma (SDPP) is an abattoir by-product used in animal nutrition with beneficial effects reported in livestock and commercial aquatic species. Previous results have found that the dietary inclusion of SDPP in gilthead sea bream (Sparus aurata) increased the density of intestinal goblet cells, and it did not result in significant changes in the autochthonous microbiota. However, there is no comprehensive data on the mechanisms that could take place on the intestine of gilthead sea bream fed with an SDPP-supplemented diet. For this reason, this study aimed to unveil the biological mechanisms modulated in response to the dietary administration of SDPP in the gilthead sea bream gut. To achieve this goal, we made a microarrays-based transcriptomic approach in gut samples from gilthead sea bream fed with an SDPP-supplemented diet for 95 days. As control diet, we used a protein-rich commercial feed (51% crude protein, 17% crude fat, and 20.6 MJ/kg gross energy) which was supplemented with 3% SDPP at the expense of LT70 fishmeal. The microarray analyses showed a total of 803 (468 up- and 335 down-regulated) differential expressed genes (DEGs). The functional network analysis revealed that dietary inclusion of SDPP induced sustained changes in 120 biological processes, grouped in 12-clusters. Among them, the metabolic-related process (cellular catabolic process, organic substance catabolic process, protein metabolism process), protein transport, and leukocyte mediated immunity interacted in the leading interactome network. This evidence confirms the previous evidence of the enhancement of the mucosal health status in response to the dietary administration of SDPP and provides further understanding of the mode of action of this ingredient in aquafeeds.info:eu-repo/semantics/publishedVersio

The Landscape of IFN/ISG Signaling in HIV-1-Infected Macrophages and Its Possible Role in the HIV-1 Latency

A key characteristic of Human immunodeficiency virus type 1 (HIV-1) infection is the generation of latent viral reservoirs, which have been associated with chronic immune activation and sustained inflammation. Macrophages play a protagonist role in this context since they are persistently infected while being a major effector of the innate immune response through the generation of type-I interferons (type I IFN) and IFN-stimulated genes (ISGs). The balance in the IFN signaling and the ISG induction is critical to promote a successful HIV-1 infection. Classically, the IFNs response is fine-tuned by opposing promotive and suppressive signals. In this context, it was described that HIV-1-infected macrophages can also synthesize some antiviral effector ISGs and, positive and negative regulators of the IFN/ISG signaling. Recently, epitranscriptomic regulatory mechanisms were described, being the N6-methylation (m6A) modification on mRNAs one of the most relevant. The epitranscriptomic regulation can affect not only IFN/ISG signaling, but also type I IFN expression, and viral fitness through modifications to HIV-1 RNA. Thus, the establishment of replication-competent latent HIV-1 infected macrophages may be due to non-classical mechanisms of type I IFN that modulate the activation of the IFN/ISG signaling network

The Analysis of Live-Attenuated Piscirickettsia salmonis Vaccine Reveals the Short-Term Upregulation of Innate and Adaptive Immune Genes in Atlantic Salmon (Salmo salar) : an in situ open-sea cages study

Piscirickettsia salmonis, the etiological agent of the Salmon Rickettsial Septicemia (SRS), is one the most serious health problems for the Chilean salmon industry. Typical antimicrobial strategies used against P. salmonis include antibiotics and vaccines, but these applications have largely failed. A few years ago, the first attenuated-live vaccine against SRS (ALPHA JECT LiVac® SRS vaccine) was released to the market. However, there is no data about the agents involved in the activation of the immune response induced under field conditions. Therefore, in this study we evaluated the expression profile of a set of gene markers related to innate and adaptive immunity in the context of a cellular response in Atlantic salmon (Salmo salar) reared under productive farm conditions and immunized with a live-attenuated vaccine against P. salmonis. We analyzed the expression at zero, 5-, 15- and 45-days post-vaccination (dpv). Our results reveal that the administration of the attenuated live SRS LiVac vaccine induces a short-term upregulation of the cellular-mediated immune response at 5 dpv modulated by the upregulation of ifnα, ifnγ, and the cd4 and cd8α T cell surface markers. In addition, we also registered the upregulation of il-10 and tgfβ. Altogether, the results suggest that a balanced activation of the immune response took place only at early times post-vaccination (5 dpv). The scope of this short-term upregulation of the cellular-mediated immune response against a natural outbreak in fish subjected to productive farm conditions deserves further research