A differential role for corticosteroid receptors in neuroendocrine-immune interactions in carp (Cyprinus carpio L.)

In this thesis we investigated the involvement of the receptors for the stress hormone cortisol in stress and immune regulation. We set out to characterise the pro-inflammatory cytokine interferon gamma (IFN-γ). Furthermore, we used a genome wide screen (microarray) to search for additional genes that might be involved in regulation of the stress or the immune response. <br/> <br/> In teleostean fishes cortisol can be bound by different receptors encoded by at least three different genes. An ancestral corticosteroid receptor (AncCR) is assumed to have been an effective receptor for cortisol in the ancestors of fishes. An early genomic duplication in the fish lineage, over 450 million years ago, led to separate glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) genes, both of which retained the ability to bind cortisol. A second major genomic duplication event took place only in teleostean fishes (not in other vertebrates), and gave rise to duplicate GR genes (GR1 and GR2). Even more variants developed as a result of alternative splicing of the GR1 gene which introduces a nine amino acid insert in the DNA-binding domain of GR1a, GR1b does not have this insert. <br/> <br/> To investigate how one ligand can regulate many and very diverse functions using multiple receptors, we describe the expression of GR1 (a and b), GR2 and MR and their sensitivity for cortisol in chapters 3 and 4. The three receptors are expressed in tissues that make up the neuroendocrine stress-axis (brain, hypothalamus and pituitary) and in cells that produce corticotropin-releasing hormone (CRH) and adrenocorticotropic hormone (ACTH). Decreased mRNA expression in brain after prolonged stress suggests an involvement in regulation of hypothalamo-pituitary-interrenal (HPI)-axis activity. In cells of the immune system MR expression is very low compared to GR expression and GR2 is preferentially expressed in lymphocytes. Transactivation assays shows that GR1 is a relatively ‘insensitive’ or ‘stress’ receptor, which can only become activated at stress levels of, whereas GR2 is a ‘sensitive’ receptor that will already be activated at basal levels of cortisol such as occur in non-stressed fish. MR sensitivity for cortisol is intermediate. We predict by tertiary protein modelling and confirmed by transfection assays, that the transactivation capacity of both splice variants (GR1a and GR1b) is similar. Based on the very low expression level in immune cells and the moderate transactivation capacity of MR we concluded that GRs rather then the MR primarily convey stress signals to the immune system. Next, we determined the expression profile of the duplicated GR genes in the immune system in chapters 4 and 5 to investigate the regulation of stress-induced immune modulation. Simultaneously we investigated the expression profile of (among others) heat shock protein 70 (Hsp70). This protein is required for binding of cortisol to the GR, but also has intrinsic immune modulatory functions, as it was shown to downregulate LPS-induced pro-inflammatory cytokine expression in vitro and in vivo. In head kidney phagocytes we found that only physiological stress levels of cortisol could reduce LPS-induced expression of pro-inflammatory cytokines, a response that appears mediated by the ‘stress’ receptor GR1. Moreover, we found that Hsp70 and GR1 (a and b) expression is increased after an immune stimulus in vitro and in vivo, whereas 24 hr restraint stress or 100nM cortisol-treatment hardly increases Hsp70 and GR1 expression levels. This suggests that an immune stimulus rather than increased cortisol levels increases the sensitivity for glucocorticoid regulation and thereby of the cytokine profiles in immune cells. <br/> <br/> To find additional genes involved in bidirectional neuroendocrine-immune communication we applied a genome wide screen of 9000 randomly picked cDNA clones. This has the advantage of an unprejudiced overview of regulated genes, but the sensitivity of the technique is limited. In chapter 6 we describe a microarray experiment in which we compared mild acute stress, to prolonged severe immune stimulation. We show that an immune response after parasite infection appears tightly regulated and comparable between individuals, whereas a mild acute stressor allows for more variable gene expression profiles. We found LOC406744 of the DUF727 protein family and nephrosin as new interesting candidate genes that may be involved in neuroendocrine-immune communication. <br/> <br/> The key pro-inflammatory cytokine IFN-γ, which is hypothesised to affect neurotransmitter and hormone release, had not been investigated in carp. In chapter 7 we show that carp have duplicate IFN-γ genes that are expressed in immune cells. IFN-γ-2 shows structural and functional characteristics simlar to those in other vertebrate IFN-γ genes and appears to be involved in T-lymphocyte function, whereas IFN-γ-1 is expressed in stimulated B-lymphocytes. Currently recombinant proteins are being produced which will enable us to further elucidate the role of both IFN-γ gene products in the immune system as well as in mediating the neuroendocrine stress response. <br/> <br/> Interestingly, as explained in chapter 8, both the glucocorticoid receptor and the IFN-γ genes are duplicated. The duplication-degeneration-complementation (DCC) model has been proposed as an explanation for the high retention of duplicate genes in fishes. The hypothesis assumes that following gene duplication, the two gene copies degenerate over time by random mutation to perform complementary functions that jointly match that of the single ancestral gene, termed ‘subfunctionalisation’. Indeed it appears that the duplicate GR genes have divided the general and ‘stress-related’ functions, reflected by their different sensitivity for cortisol. The duplicate IFN-γ genes appear to have divided B- and T-lymphocyte functions as targets suggested by their gene expression profiles upon selective stimulation. <br/> <br/> An important conclusion of this thesis is that duplicated glucocorticoid receptors and heat shock proteins are an integral part of the immune system. Immune stimuli rather than increased cortisol levels control GR and Hsp70 expression in immune cells. The differentially regulated expression of GR genes is at the basis of a balanced pro- and anti-inflammatory <br/> cytokine profile, immune cell viability and thus at the basis of the success of the fishes. This thesis illustrates the importance of extensive and effective bidirectional communication between the neuroendocrine and immune systems, which are at the basis of the successful evolution of the vertebrates.