Gene expression profiles in Atlantic salmon adipose-derived stromo-vascular fraction during differentiation into adipocytes

<p>Abstract</p> <p>Background</p> <p>Excessive fat deposition is one of the largest problems faced by salmon aquaculture industries, leading to production losses due to high volume of adipose tissue offal. In addition, increased lipid accumulation may impose considerable stress on adipocytes leading to adipocyte activation and production and secretion of inflammatory mediators, as observed in mammals.</p> <p>Results</p> <p>Microarray and qPCR analyses were performed to follow transcriptome changes during adipogenesis in the primary culture of adipose stromo-vascular fraction (aSVF) of Atlantic salmon. Cellular heterogeneity decreased by confluence as evidenced by the down-regulation of markers of osteo/chondrogenic, myogenic, immune and vasculature lineages. Transgelin (TAGLN), a marker of the multipotent pericyte, was prominently expressed around confluence while adipogenic PPARγ was up-regulated already in subconfluent cells. Proliferative activity and subsequent cell cycle arrest were reflected in the fluctuations of pro- and anti-mitotic regulators. Marked regulation of genes involved in lipid and glucose metabolism and pathways producing NADPH and glycerol-3-phosphate (G3P) was seen during the terminal differentiation, also characterised by diverse stress responses. Activation of the glutathione and thioredoxin antioxidant systems and changes in the iron metabolism suggested the need for protection against oxidative stress. Signs of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) occured in parallel with the increased lipid droplet (LD) formation and production of secretory proteins (adipsin, visfatin). The UPR markers XBP1 and ATF6 were induced together with genes involved in ubiquitin-proteasome and lysosomal proteolysis. Concurrently, translation was suppressed as evidenced by the down-regulation of genes encoding elongation factors and components of the ribosomal machinery. Notably, expression changes of a panel of genes that belong to different immune pathways were seen throughout adipogenesis. The induction of AP1 (Jun, Fos), which is a master regulator of stress responses, culminated by the end of adipogenesis, concurrent with the maximal observed lipid deposition.</p> <p>Conclusions</p> <p>Our data point to an intimate relationship between metabolic regulation and immune responses in white adipocytes of a cold-blooded vertebrate. Stress imposed on adipocytes by LD formation and expansion is prominently reflected in the ER compartment and the activated UPR response could have an important role at visceral obesity in fish.</p

Hepatic gene expression profiling reveals protective responses in Atlantic salmon vaccinated against furunculosis

<p>Abstract</p> <p>Background</p> <p>Furunculosis, a disease caused with gram negative bacteria <it>Aeromonas salmonicida </it>produces heavy losses in aquaculture. Vaccination against furunculosis reduces mortality of Atlantic salmon but fails to eradicate infection. Factors that determine high individual variation of vaccination efficiency remain unknown. We used gene expression analyses to search for the correlates of vaccine protection against furunculosis in Atlantic salmon.</p> <p>Results</p> <p>Naïve and vaccinated fish were challenged by co-habitance. Fish with symptoms of furunculosis at the onset of mass mortality (LR - low resistance) and survivors (HR - high resistance) were sampled. Hepatic gene expression was analyzed with microarray (SFA2.0 - immunochip) and real-time qPCR. Comparison of LR and HR indicated changes associated with the protection and results obtained with naïve fish were used to find and filter the vaccine-independent responses. Genes involved in recruitment and migration of immune cells changed expression in both directions with greater magnitude in LR. Induction of the regulators of immune responses was either equal (NFkB) or greater (Jun) in LR. Expression levels of proteasome components and extracellular proteases were higher in LR while protease inhibitors were up-regulated in HR. Differences in chaperones and protein adaptors, scavengers of reactive oxygen species and genes for proteins of iron metabolism suggested cellular and oxidative stress in LR. Reduced levels of free iron and heme can be predicted in LR by gene expression profiles with no protection against pathogen. The level of complement regulation was greater in HR, which showed up-regulation of the components of membrane attack complex and the complement proteins that protect the host against the auto-immune damages. HR fish was also characterized with up-regulation of genes for proteins involved in the protection of extracellular matrix, lipid metabolism and clearance of endogenous and exogenous toxic compounds. A number of genes with marked expression difference between HR and LR can be considered as positive and negative correlates of vaccine protection against furunculosis.</p> <p>Conclusion</p> <p>Efficiency of vaccination against furunculosis depends largely on the ability of host to neutralize the negative impacts of immune responses combined with efficient clearance and prevention of tissue damages.</p

Genomiske responser på patogene agens og foringsmessige intervensjoner hos salmonide

Aquaculture exposes salmonid fish to diverse stressors, including pathogens and inadequate nutrition. The identification of molecular basis underlying compromised health and growth caused by these factors is confounded by the complexity of induced responses and simultaneous action of multiple stressors. The goal of this thesis was to improve knowledge of several adverse conditions by means of functional genomics. The molecular processes associated with disease resistance and fast growth were investigated with an aid of the salmonid cDNA microarray (SFA.2) enriched with genes involved in stress and immune responses. Genome-wide gene expression profiling was followed with qPCR confirmation of the most important findings. A similar design of experiments was applied. The selection of organs for analyses was based on pilot microarray screening that detected the most informative responses from a wider range of tissue samples. The microarray and qPCR analyses of the transcriptomes revealed the gene expression changes behind the studied phenotypes. Paper I addressed the molecular determinants underlying high susceptibility to sea lice in Atlantic salmon. Systemic nature of responses and previously unknown role for adaptive immunity were revealed. Alternatively activated macrophages and predominant type 2 differentiation of T helper (Th) cells were observed in response to the parasite. However, this early activation was followed by general immunosuppression in all organs while cellular stress markedly increased in damaged skin. Further, the observed gene expression profile supported the notion of delayed healing of wounds inflicted by sea lice. Vaccine-dependent determinants of protective responses in Atlantic salmon against the causative agent of furunculosis, Aeromonas salmonicida were investigated in Paper II. The results suggested that tight regulation of the inflammatory response is beneficial for the host. The selective induction of complement components in vaccinated survivors was the only observed enhanced effector immune mechanism. Importantly, elevated expression of genes involved in repair and prevention of immune-mediated damages and in clearance of endogenous and exogenous toxic compounds were associated with survival. In Paper III, the ability of β-glucan lentinan from shiitake mushroom to modulate inflammation induced by injected lipopolysaccharide (LPS) was tested in rainbow trout. Lentinan dampened the expression of numerous proinflammatory genes in spleen whose activation could have detrimental consequences for the host. Interestingly, cross-experimental comparison revealed remarkable similarities between responses of lentinan fed fish and those observed in salmon resistant to A. salmonicida (Paper II) and infectious salmon anemia virus (ISAV) [1]. Thus, avoiding immunopathology by selective suppression of potentially harmful immune responses was predicted for LPS-challenged trout pre-conditioned with lentinan. Paper IV examined the effects of three dietary regimens based on: 1) partial fish meal replacement by extracted soybean meal, 2) feeding of control diet at reduced ration and 3) the combination of the interventions, feeding of soybean meal based diet at reduced ration. Highly similar transcriptional responses to soy and reduced feeding were revealed while the joint treatment unexpectedly resulted in weaker expression changes of the majority of hepatic genes. The observed changes in a number of metabolic pathways indicated a high level of conservation of responses to restricted feeding between the cold-blooded and warm-blooded vertebrates. The study identified twelve nutrition-responsive candidate biomarkers but no specific differences between groups. Slower growth could be beneficial for health of salmon due to down-regulation of genes involved in inflammation and stress responses. The performed studies indicated the regulations of gene expression that can determine the ability of fish cells and tissues to sense and adapt to a wide range of challenges. The identified differentially expressed genes are involved in both generalized and specific responses. Notably, the results illustrate highly contextual nature of biological responses exemplified by inflammation, which is regarded as a “double-edged sword”. Th2-modified response was linked to high susceptibility to sea lice in salmon (Paper I) while fish with excessive inflammatory responses succumbed to furunculosis (Paper II). Results from Paper III were in line with these findings; detrimental consequences from LPS exposure due to overly activated inflammation could be predicted for fish not fed with lentinan. The three studies with pathogenic agents thus illustrate the paramount importance of properly balanced immune responses and their subsequent regulation. Slower growth might be favourable in this sense, as the attenuated gene expression of cellular stress markers and potentially dangerous inflammatory mediators was shown in Paper IV.Norges Forskningsrå