Inflammatory responses in primary muscle cell cultures in Atlantic salmon (Salmo salar)

Abstract

Background: The relationship between fish health and muscle growth is critical for continued expansion of the aquaculture industry. The effect of immune stimulation on the expression of genes related to the energy balance of fish is poorly understood. In mammals immune stimulation results in major transcriptional changes in muscle, potentially to allow a reallocation of amino acids for use in the immune response and energy homeostasis. The aim of this study was to investigate the effects of immune stimulation on fish muscle gene expression.

Results: Atlantic salmon (Salmo salar) primary muscle cell cultures were stimulated with recombinant (r)IL-1β, a major proinflammatory cytokine, for 24 h in order to simulate an acute immune response. The transcriptomic response was determined by RNA hybridization to a 4 × 44 K Agilent Atlantic salmon microarray platform. The rIL-1β stimulation induced the expression of genes related to both the innate and adaptive immune systems. In addition there were highly significant changes in the expression of genes related to regulation of the cell cycle, growth/structural proteins, proteolysis and lipid metabolism. Of interest were a number of IGF binding proteins that were differentially expressed, which may demonstrate cross talk between the growth and immune systems.

Conclusion: We show rIL-1β modulates the expression of not only immune related genes, but also that of genes involved in processes related to growth and metabolism. Co-stimulation of muscle cells with both rIGF-I and rIL-1β demonstrates cross talk between these pathways providing potential avenues for further research. This study highlights the potential negative effects of inflammation on muscle protein deposition and growth in fish and extends our understanding of energy allocation in ectothermic animals.

Figure 1

Bar chart showing the 15 Gene ontologies found to be most highly statistically enriched in response to rIL-1β stimulation of muscle cells in vitro. Gene ontology enrichment carried out using GOEAST, GOslimming of the subsequent list performed with REVIGO.

Figure 2

Graph showing the fold effects of rIL-1β stimulation compared to control on the expression of genes involved in the immune response and growth after 6, 24 and 48 h stimulation. Statistics were carried out using a one way ANOVA. Time points that do not share a letter are statistically different from each other. All mRNAs examined here were significantly altered in expression at all time points relative to the unstimulated control.

Figure 3

Fold change of genes involved in both the immune response and growth in response to 6 h stimulation with either rIL-1β (25 ng/ml), rIL-1β (25 ng/ml) + rIGF(100nM), or rIGF(100nM). * represents a significant difference from control, bars which share a letter are not significantly different. All fold changes were calculated compared to unstimulated control samples. Comparative gene expression was measured with qPCR.

Figure 4

Fold change of genes involved in both the immune response and growth in response to 24 h stimulation with either rIL-1β (25 ng/ml), rIL-1β (25 ng/ml) + rIGF(100nM), or rIGF(100nM). * represents a significant difference from control, bars which share a letter are not significantly different. All fold changes were calculated compared to unstimulated control samples. Comparative gene expression was measured with qPCR.

Figure 5

Model of the proposed actions of rIL-1β on gene expression and physiology of muscle cells in vitro. rIL-1β is recognised by its receptor and an accessory protein, this then sets off a signal cascade resulting in the activation of NFκβ and JUN which in turn result in changes in gene expression and physiology. The proposed changes in physiology are shown in black. Green arrows show a positive effect on this aspect of physiology and red arrows show a negative effect. A sample of genes related to each aspect of physiology is shown in either red (up regulation) or green (down regulation).

Figure 6

The experimental design carried out for the microarray experiment. Cells were genereated from six individual fish, these cells were pooled and plated onto six well plates. RNA extracted from four biological replicates stimulated with rIL-1β stimulation were kept separate. For control wells, RNA was pooled to generate a common reference RNA sample.