Effect of yeast (Xanthophyllomyces dendrorhous) and plant (Saint John's wort, lemon balm, and rosemary) extract based functional diets on antioxidant and immune status of Atlantic salmon (Salmo salar) subjected to crowding stress.

Salmon farming may face stress due to the intensive culture conditions with negative impacts on overall performance. In this aspect, functional feed improves not only the basic nutritional requirements but also the health status and fish growth. However, to date no studies have been carried out to evaluate the effect of functional diets in salmon subjected to crowding stress. Thus, the aim of this study was to evaluate the effect of yeast extract (Xanthophyllomyces dendrorhous; diet A) and the combination of plant extracts (common Saint John's wort, lemon balm, and rosemary; diet B) on the antioxidant and immune status of Atlantic salmon grown under normal cultured conditions and then subjected to crowding stress. Fish were fed with functional diets during 30 days (12 kg/m3) and then subjected to crowding stress (20 kg/m3) for 10 days. The lipid peroxidation in gut showed that both diets induced a marked decrease on oxidative damage when fish were subjected to crowding stress. The protein carbonylation in muscle displayed at day 30 a marked decrease in both functional diets that was more marked on the stress condition. The expression of immune markers (IFNγ, CD4, IL-10, TGF-ß, IgMmb, IgMsec, T-Bet, and GATA-3) indicated the upregulation of those associated to humoral-like response (CD4, IL-10, GATA-3) when fish were subjected to crowding stress. These results were confirmed with the expression of secreted IgM. Altogether, these functional diets improved the antioxidant status and increased the expression of genes related to Th2-like response suggesting a protective role on fish subjected to crowding stress.

KLF17 is an important regulatory component of the transcriptomic response of Atlantic salmon macrophages to Piscirickettsia salmonis infection.

Piscirickettsia salmonis is the most important health problem facing Chilean Aquaculture. Previous reports suggest that P. salmonis can survive in salmonid macrophages by interfering with the host immune response. However, the relevant aspects of the molecular pathogenesis of P. salmonis have been poorly characterized. In this work, we evaluated the transcriptomic changes in macrophage-like cell line SHK-1 infected with P. salmonis at 24- and 48-hours post-infection (hpi) and generated network models of the macrophage response to the infection using co-expression analysis and regulatory transcription factor-target gene information. Transcriptomic analysis showed that 635 genes were differentially expressed after 24- and/or 48-hpi. The pattern of expression of these genes was analyzed by weighted co-expression network analysis (WGCNA), which classified genes into 4 modules of expression, comprising early responses to the bacterium. Induced genes included genes involved in metabolism and cell differentiation, intracellular transportation, and cytoskeleton reorganization, while repressed genes included genes involved in extracellular matrix organization and RNA metabolism. To understand how these expression changes are orchestrated and to pinpoint relevant transcription factors (TFs) controlling the response, we established a curated database of TF-target gene regulatory interactions in Salmo salar, SalSaDB. Using this resource, together with co-expression module data, we generated infection context-specific networks that were analyzed to determine highly connected TF nodes. We found that the most connected TF of the 24- and 48-hpi response networks is KLF17, an ortholog of the KLF4 TF involved in the polarization of macrophages to an M2-phenotype in mammals. Interestingly, while KLF17 is induced by P. salmonis infection, other TFs, such as NOTCH3 and NFATC1, whose orthologs in mammals are related to M1-like macrophages, are repressed. In sum, our results suggest the induction of early regulatory events associated with an M2-like phenotype of macrophages that drives effectors related to the lysosome, RNA metabolism, cytoskeleton organization, and extracellular matrix remodeling. Moreover, the M1-like response seems delayed in generating an effective response, suggesting a polarization towards M2-like macrophages that allows the survival of P. salmonis. This work also contributes to SalSaDB, a curated database of TF-target gene interactions that is freely available for the Atlantic salmon community.

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.