Stress-Immune-Growth Interactions: Cortisol Modulates Suppressors of Cytokine Signaling and JAK/STAT Pathway in Rainbow Trout Liver

<div><p>Chronic stress is a major factor in the poor growth and immune performance of salmonids in aquaculture. However, the molecular mechanisms linking stress effects to growth and immune dysfunction is poorly understood. The suppressors of cytokine signaling (SOCS), a family of genes involved in the inhibition of JAK/STAT pathway, negatively regulates growth hormone and cytokine signaling, but their role in fish is unclear. Here we tested the hypothesis that cortisol modulation of SOCS gene expression is a key molecular mechanism leading to growth and immune suppression in response to stress in fish. Exposure of rainbow trout (<i>Oncorhynchus mykiss</i>) liver slices to cortisol, mimicking stress level, upregulated SOCS-1 and SOCS-2 mRNA abundance and this response was abolished by the glucocorticoid receptor antagonist mifepristone. Bioinformatics analysis confirmed the presence of putative glucocorticoid response elements in rainbow trout SOCS-1 and SOCS-2 promoters. Prior cortisol treatment suppressed acute growth hormone (GH)-stimulated IGF-1 mRNA abundance in trout liver and this involved a reduction in STAT5 phosphorylation and lower total JAK2 protein expression. Prior cortisol treatment also suppressed lipopolysaccharide (LPS)-induced IL-6 but not IL-8 transcript levels; the former but not the latter cytokine expression is via JAK/STAT phosphorylation. LPS treatment reduced GH signaling, but this was associated with the downregulation of GH receptors and not due to the upregulation of SOCS transcript levels by this endotoxin. Collectively, our results suggest that upregulation of SOCS-1 and SOCS-2 transcript levels by cortisol, and the associated reduction in JAK/STAT signaling pathway, may be a novel mechanism leading to growth reduction and immune suppression during stress in trout.</p></div

Pre-exposure to cortisol and LPS affect GH signaling.

<p>The graphs show the effects of cortisol and LPS either singly or in combination in modulating GH stimulation of IGF-1 mRNA abundance (A), STAT5 phosphorylation (B) and total JAK2 protein expression (C) in rainbow trout liver. Rainbow trout liver slices were pre-incubated with control media or media containing cortisol (100 ng/ml; Sigma), LPS (30 μg/ml) or a combination of cortisol and LPS for 24 h, after which they were incubated with or without GH (500 ng/ml) for either 10 min (JAK/STAT) or 6 h (IGF-1). Values are plotted as % no GH control and show mean ± S.E.M (n = 6 fish livers); different lower case letters denote significant treatment effects and interations; * denotes overall GH effects; the inset shows overall cortisol effects (two way repeated measures ANOVA, p < 0.05).</p

Cortisol upregulates SOCS mRNA abundance.

<p>The effect of cortisol on the temporal profiles of SOCS-1 (A) and SOCS-2 (B) mRNA abundance in rainbow trout liver. Liver slices were incubated with either control media or media containing cortisol (100 ng/ml) for 1, 4, 6, 8 and 24 h. Values are plotted as % control and show mean ± S.E.M (n = 6 fish livers); different lower case letters denote significant treatment effects within each timepoint; (two way ANOVA, p < 0.05).</p

Pre-exposure to cortisol suppresses acute GH signaling.

<p>The effect of cortisol and GH on IGF-1 mRNA abundance (A), STAT5 phosphorylation (B) and total JAK2 protein expression (C) in rainbow trout liver. Liver slices were pre-incubated with cortisol (100ng/ml; Sigma) or control media for 24 h and then stimulated with GH (100ng/ml or 1000ng/ml) for either 10 min (JAK/STAT) or 6 h (IGF-1). Values are plotted as % no cortisol control and shown as mean ± S.E.M (n = 6 fish livers); different lower case letters denote significant treatment effects and interactions; different upper case letters denote overall GH effects between the control, 100 GH and 1000 GH groups; the inset shows overall cortisol effects (two way repeated measures ANOVA, p < 0.05).</p

Gene-specific primers for quantitative real-time PCR.

<p>List of genes (Gene ID), forward and reverse primer sequences, annealing temperature and amplicon size. IL-1β: interleukin-1 beta; IL-8: interleukin-8; IL-6: interleukin-6; SOCS: suppressors of cytokine signaling; IGF-1: insulin like growth factor-1; GHR1: growth hormone receptor-1; GHR2: growth hormone receptor-2; EF1α: elongation factor 1α.</p><p>Gene-specific primers for quantitative real-time PCR.</p

A proposed model for cortisol effect on growth and immune suppression in trout liver.

<p>Stressed levels of cortisol elevate SOCS transcript levels and reduce GH signaling and the corresponding IGF-1 expression in rainbow trout liver by preventing STAT5 phosphorylation and decreasing total JAK2 protein expression. The cortisol-induced upregulation of SOCS may be playing a role in the suppression of LPS-induced IL-6 expression (a cytokine signaling through the JAK/STAT pathway). Immune challenge with LPS may indirectly inhibit GH signaling by elevating plasma cortisol levels or directly inhibit GH signaling and the corresponding IGF-1 expression by downregulating growth hormone receptors 1 and 2 and by preventing STAT5 phosphorylation.</p

Effect of cortisol and LPS on GH receptors.

<p>The effect of cortisol and LPS on GHR1 (A) and GHR2 (B) mRNA abundance in rainbow trout liver. Rainbow trout liver slices were pre-incubated with control media or media containing cortisol (100 ng/ml; Sigma), LPS (30 μg/ml) or a combination of cortisol and LPS for 24 h. Values are plotted as % control and show mean ± S.E.M (n = 6 fish livers); different lower case letters denote significant treatment effects; * denotes overall cortisol effects (two way repeated measures ANOVA, p < 0.05).</p

Glucocorticoid receptor signaling is involved in SOCS upregulation.

<p>The effect of cortisol and mifepristone either alone or in combination on SOCS-1 (A) and SOCS-2 (B) mRNA abundance in rainbow trout liver. Liver slices were incubated with control media or media containing cortisol (100 ng/ml), mifepristone (MP; 1000 ng/mL; Sigma) or a combination of mifepristone and cortisol for 24 h. Values are plotted as % control and show mean ± S.E.M (n = 6 fish livers); different upper case letters denote significant treatment effects (One way repeated measures ANOVA, p < 0.05).</p

Pre-exposure to cortisol suppresses LPS signaling.

<p>Cortisol modulates LPS-induced IL-6 (A) but not IL-8 (B) mRNA abundance in rainbow trout liver. This is paralleled by an increase in SOCS-2 expression with cortisol exposure (C). Liver slices were pre-incubated with control media or media containing cortisol (100ng/ml) for 24 h after which they were incubated with control media or media containing LPS (30μg/ml) for 6 h. Values are plotted as % control and shown as mean ± S.E.M (n = 7 fish livers); different lower case letters denote significant treatment effects and interactions; * denotes overall cortisol effects (two way repeated measures ANOVA, p < 0.05).</p