The importance of CXC-receptors CXCR1-2 and CXCR4 for adaptive regulation of the stress axis in teleost fish.

In an ever-changing environment, an adaptive stress response is the pivotal regulatory mechanism to maintain allostasis. Physiologic responses to stressors enable to overcome potential threat. Glucocorticoid effects can be considered compensatory and adaptive, however prolonged or excessive glucocorticoid secretion can be also maladaptive and detrimental. Therefore, it must be tightly regulated. Apart from the essential hormonal feedback regulation, evidence accrues that cytokines, e.g., proinflammatory interleukin 1ß (IL-1ß), also play an important regulatory role in the stress axis. Here we focused on the potential role of CXC chemokines (CXCL8 and CXCL12) and their receptors (CXCR1, 2 and 4) in the regulation of the stress response in common carp. We studied changes in gene expression of CXC chemokines and CXCRs in the stress axis organs (hypothalamus-pituitary gland-head kidney) upon 11 h of restraint stress and we established how CXCR blocking affects the activation of the stress axis and the synthesis/conversion of cortisol. During restraint stress, gene expression of the majority of the proinflammatory CXCL8 and homeostatic CXCL12 chemokines and their receptors was upregulated in the stress axis organs. Inhibition of CXCR1-2 and CXCR4 differentially affected the expression of genes encoding stress-related molecules: hormones, binding proteins, receptors as well as expression of genes encoding IL-1ß and its receptor. Moreover, we observed that CXC chemokines, via interaction with their respective CXCRs, regulate gene expression of molecules involved in cortisol synthesis and conversion and consistently affect the level of cortisol released into the circulation during the stress response. We revealed that in fish, CXC chemokines and their receptors are important regulators of the stress response at multiple levels of the stress axis, with particularly pronounced effects on steroidogenesis and cortisol conversion in the head kidney.

17α-ethinylestradiol and 4-tert-octylphenol concurrently disrupt the immune response of common carp.

The aquatic environment is massively polluted with endocrine-disrupting compounds (EDCs) including synthetic estrogens (e.g. 17α-ethinylestradiol, EE2) and alkylphenols (e.g. 4-tert-octylphenol, 4t-OP). A major mechanism of action for estrogenic EDCs is their interaction with estrogen receptors and consequently their modulation of the action of enzymes involved in steroid conversion e.g. aromatase CYP19. We now studied the effects of EE2 and 4t-OP on the anti-bacterial immune response of common carp. We investigated effects on the number/composition of inflammatory leukocytes and on the gene expression of mediators that regulate inflammation and EDC binding. In vitro we found that high concentrations of both EE2 and 4t-OP down-regulated IFN-γ2 and IFN-γ-dependent immune responses in LPS-stimulated monocytes/macrophages. Similarly, during bacterial infection in fish, in vivo treated with EE2 and 4t-OP, decreased gene expression of il-12p35 and of ifn-γ2 was found in the focus of inflammation. Moreover, during A. salmonicida-induced infection in EE2-treated carp, but not in fish fed with 4t-OP-treated food, we found an enhanced inflammatory reaction manifested by high number of inflammatory peritoneal leukocytes, including phagocytes and higher expression of pro-inflammatory mediators (inos, il-1ß, cxcl8_l2). Furthermore, in the liver, EE2 down-regulated the expression of acute phase proteins: CRPs and C3. Importantly, both in vitro and in vivo, EDCs altered the expression of estrogen receptors: nuclear (erα and erß) and membrane (gpr30). EDCs also induced up-regulation of the cyp19b gene. Our findings reveal that contamination of the aquatic milieu with estrogenic EDCs, may considerably violate the subtle and particular allostatic interactions between the immune response and endogenous estrogens and this may have negative consequences for fish health.

Cortisol Metabolism in Carp Macrophages: A Role for Macrophage-Derived Cortisol in M1/M2 Polarization.

Macrophages are crucial not only for initiation of inflammation and pathogen eradication (classically polarized M1 macrophages), but also for inflammation inhibition and tissue regeneration (alternatively polarized M2 macrophages). Their polarization toward the M1 population occurs under the influence of interferon-γ + lipopolysaccharide (IFN-γ + LPS), while alternatively polarized M2 macrophages evolve upon, e.g., interlukin 4 (IL-4) or cortisol stimulation. This in vitro study focused on a possible role for macrophage-derived cortisol in M1/M2 polarization in common carp. We studied the expression of molecules involved in cortisol synthesis/conversion from and to cortisone like 11ß-hydroxysteroid dehydrogenase type 2 and 3. (11ß-HSD2 and 3) and 11ß-hydroxylase (CYP11b), as well as the expression of glucocorticoid receptors (GRs) and proliferator-activated receptor gamma (PPARγ) in M1 and M2 macrophages. Lastly, we analyzed how inhibition of these molecules affect macrophage polarization. In M1 cells, upregulation of gene expression of GRs and 11ß-HSD3 was found, while, in M2 macrophages, expression of 11ß-hsd2 was upregulated. Moreover, blocking of cortisol synthesis/conversion and GRs or PPARγ induced changes in expression of anti-inflammatory interleukin 10 (IL-10). Consequently, our data show that carp monocytes/macrophages can convert cortisol. The results strongly suggest that cortisol, via intracrine interaction with GRs, is important for IL-10-dependent control of the activity of macrophages and for the regulation of M1/M2 polarization to finally determine the outcome of an infection.

Effects of stress and cortisol on the polarization of carp macrophages.

In teleost fish, myelopoiesis is maintained both in the head (HK) and trunk kidney (TK), but only the HK holds the endocrine cells that produce the stress hormone cortisol. We now compared the effects of prolonged restraint stress (in vivo) and cortisol (in vitro) on the polarization of HK and TK-derived carp macrophages. Monocytes/macrophages from both sources were treated in vitro with cortisol, lipopolysaccharide or with both factors combined. In vivo, fish were challenged by a prolonged restraint stress. Gene expression of several markers typical for classical M1 and alternative M2 macrophage polarization, as well as glucocorticoid receptors, were measured. Cells from both sources did not differ in the constitutive gene expression of glucocorticoid receptors, whereas they significantly differed in their response to cortisol and stress. In the LPS-stimulated HK monocytes/macrophages, cortisol in vitro counteracted the action of LPS while the effects of cortisol on the activity of TK monocytes/macrophages were less explicit. In vivo, restraint stress up-regulated gene expression of M2 markers in freshly isolated HK monocytes/macrophages, while at the same time it did not affect TK monocytes/macrophages. Moreover, LPS-stimulated HK monocytes/macrophages from stressed animals showed only minor differences in the gene expression of M1 and M2 markers, compared to LPS-treated monocytes/macrophages from control fish. In contrast, stress-induced changes in TK-derived LPS-treated cells were more pronounced. However, these changes did not clearly indicate whether in TK monocytes/macrophages stress will stimulate classical or alternative polarization. Altogether, our results imply that cortisol in vitro and stress in vivo direct HK, but not TK, monocytes/macrophages to the path of alternative polarization. These findings reveal that like in mammals, also in fish the glucocorticoids form important stimulators of alternative macrophage polarization.

A role for CXC chemokines and their receptors in stress axis regulation of common carp.

Although chemokines mainly function to activate leukocytes and to direct their migration, novel evidence indicates non-immune functions for chemokines within the nervous and endocrine systems. These include development of the nervous system, neuromodulation, neuroendocrine regulation and direct neurotransmitter-like actions. In order to clarify a potential role for chemokines and their receptors in the stress response of fish, we studied changes in the expression patterns of CXC ligands and their receptors in the stress axis organs of carp, during a restraint stress procedure. We showed that stress down-regulated the gene expression of CXCL9-11 (CXCb1 and CXCb2) in stress axis organs and up-regulated expression of CXCR4 chemokine receptor in NPO and pituitary. Moreover, upon stress, reduced gene expression of CXCL12a and CXCL14 was observed in the head kidney. Our results imply that in teleost fish, CXC chemokines and their receptors are involved in neuroendocrine regulation. The active regulation of their expression in stress axis organs during periods of restraint indicates a significant role in the stress response.

Estrogen-dependent seasonal adaptations in the immune response of fish.

Clinical and experimental evidence shows that estrogens affect immunity in mammals. Less is known about this interaction in the evolutionary older, non-mammalian, vertebrates. Fish form an excellent model to identify evolutionary conserved neuroendocrine-immune interactions: i) they are the earliest vertebrates with fully developed innate and adaptive immunity, ii) immune and endocrine parameters vary with season, and iii) physiology is constantly disrupted by increasing contamination of the aquatic environment. Neuro-immuno-endocrine interactions enable adaption to changing internal and external environment and are based on shared signaling molecules and receptors. The presence of specific estrogen receptors on/in fish leukocytes, implies direct estrogen-mediated immunoregulation. Fish leukocytes most probably are also capable to produce estrogens as they express the cyp19a and cyp19b - genes, encoding aromatase cytochrome P450, the enzyme critical for conversion of C19 steroids to estrogens. Immunoregulatory actions of estrogens, vary among animal species, and also with dose, target cell type, or physiological condition (e.g., infected/non-infected, reproductive status). They moreover are multifaceted. Interestingly, season-dependent changes in immune status correlate with changes in the levels of circulating sex hormones. Whereas E2 circulating in the bloodstream is perhaps the most likely candidate to be the physiological mediator of systemic immune-reproductive trade-offs, leukocyte-derived hormones are hypothesized to be mainly involved in local tuning of the immune response. Contamination of the aquatic environment with estrogenic EDCs may violate the delicate and precise allostatic interactions between the endogenous estrogen system and the immune system. This has negative effects on fish health, but will also affect the physiology of its consumers.

Stress differentially affects the systemic and leukocyte estrogen network in common carp.

Both systemic and locally released steroid hormones, such as cortisol and estrogens, show immunomodulatory actions. This research gives evidence that circulating and leukocyte-derived estrogens can be involved in the regulation of the immune response in common carp, during homeostasis and upon restraining stress. It was found that stress reduced level of blood 17ß-estradiol (E2) and down-regulated the gene expression of components of the "classical" estrogen system: the nuclear estrogen receptors and the aromatase CYP19, in the hypothalamus, the pituitary and in the ovaries. In contrast, higher gene expression of the nuclear estrogen receptors and cyp19a was found in the head kidney of stressed animals. Moreover, stress induced changes in the E2 level and in the estrogen sensitivity at local/leukocyte level. For the first time in fish, we showed the presence of physiologically relevant amounts of E2 and the substrates for its conversion (estrone - E1 and testosterone - T) in head kidney monocytes/macrophages and found that its production is modulated upon stress. Moreover, stress reduced the sensitivity of leukocytes towards estrogens, by down-regulation the expression of the erb and cyp19 genes in carp phagocytes. In contrast, era expression was up-regulated in the head kidney monocytes/macrophages and in PBLs derived from stressed animals. We hypothesize that, the increased expression of ERα, that was observed during stress, can be important for the regulation of leukocyte differentiation, maturation and migration. In conclusion, these results indicate that, in fish, the estrogen network can be actively involved in the regulation of the systemic and local stress response and the immune response.

A short-term extremely low frequency electromagnetic field exposure increases circulating leukocyte numbers and affects HPA-axis signaling in mice.

There is still uncertainty whether extremely low frequency electromagnetic fields (ELF-EMF) can induce health effects like immunomodulation. Despite evidence obtained in vitro, an unambiguous association has not yet been established in vivo. Here, mice were exposed to ELF-EMF for 1, 4, and 24 h/day in a short-term (1 week) and long-term (15 weeks) set-up to investigate whole body effects on the level of stress regulation and immune response. ELF-EMF signal contained multiple frequencies (20-5000 Hz) and a magnetic flux density of 10 µT. After exposure, blood was analyzed for leukocyte numbers (short-term and long-term) and adrenocorticotropic hormone concentration (short-term only). Furthermore, in the short-term experiment, stress-related parameters, corticotropin-releasing hormone, proopiomelanocortin (POMC) and CYP11A1 gene-expression, respectively, were determined in the hypothalamic paraventricular nucleus, pituitary, and adrenal glands. In the short-term but not long-term experiment, leukocyte counts were significantly higher in the 24 h-exposed group compared with controls, mainly represented by increased neutrophils and CD4 ± lymphocytes. POMC expression and plasma adrenocorticotropic hormone were significantly lower compared with unexposed control mice. In conclusion, short-term ELF-EMF exposure may affect hypothalamic-pituitary-adrenal axis activation in mice. Changes in stress hormone release may explain changes in circulating leukocyte numbers and composition. Bioelectromagnetics. 37:433-443, 2016. © 2016 The Authors. Bioelectromagnetics Published by Wiley Periodicals, Inc.

Calcium signalling in human neutrophil cell lines is not affected by low-frequency electromagnetic fields.

We are increasingly exposed to low-frequency electromagnetic fields (LF EMFs) by electrical devices and power lines, but if and how these fields interact with living cells remains a matter of debate. This study aimed to investigate the potential effect of LF EMF exposure on calcium signalling in neutrophils. In neutrophilic granulocytes, activation of G-protein coupled receptors leads to efflux of calcium from calcium stores and influx of extracellular calcium via specialised calcium channels. The cytoplasmic rise of calcium induces cytoskeleton rearrangements, modified gene expression patterns, and cell migration. If LF EMF modulates intracellular calcium signalling, this will influence cellular behaviour and may eventually lead to health problems. We found that calcium mobilisation upon chemotactic stimulation was not altered after a short 30 min or long-term LF EMF exposure in human neutrophil-like cell lines HL-60 or PLB-985. Neither of the two investigated wave forms (Immunent and 50 Hz sine wave) at three magnetic flux densities (5 µT, 300 µT, and 500 µT) altered calcium signalling in vitro. Gene-expression patterns of calcium-signalling related genes also did not show any significant changes after exposure. Furthermore, analysis of the phenotypical appearance of microvilli by scanning electron microscopy revealed no alterations induced by LF EMF exposure. The findings above indicate that exposure to 50 Hz sinusoidal or Immunent LF EMF will not affect calcium signalling in neutrophils in vitro.

Evolutionarily conserved mechanisms regulating stress-induced neutrophil redistribution in fish.

Introduction: Stress may pose a serious challenge to immune homeostasis. Stress however also may prepare the immune system for challenges such as wounding or infection, which are likely to happen during a fight or flight stress response. Methods: In common carp (Cyprinus carpio L.) we studied the stress-induced redistribution of neutrophils into circulation, and the expression of genes encoding CXC chemokines known to be involved in the regulation of neutrophil retention (CXCL12) and redistribution (CXCL8), and their receptors (CXCR4 and CXCR1-2, respectively) in blood leukocytes and in the fish hematopoietic organ - the head kidney. The potential involvement of CXC receptors and stress hormone receptors in stress-induced neutrophil redistribution was determined by an in vivo study with selective CXCR inhibitors and antagonists of the receptors involved in stress regulation: glucocorticoid/mineralocorticoid receptors (GRs/MRs), adrenergic receptors (ADRs) and the melanocortin 2 receptor (MC2R). Results: The stress-induced increase of blood neutrophils was accompanied by a neutrophil decrease in the hematopoietic organs. This increase was cortisol-induced and GR-dependent. Moreover, stress upregulated the expression of genes encoding CXCL12 and CXCL8 chemokines, their receptors, and the receptor for granulocytes colony-stimulation factor (GCSFR) and matrix metalloproteinase 9 (MMP9). Blocking of the CXCR4 and CXCR1 and 2 receptors with selective inhibitors inhibited the stress-induced neutrophil redistribution and affected the expression of genes encoding CXC chemokines and CXCRs as well as GCSFR and MMP9. Discussion: Our data demonstrate that acute stress leads to the mobilization of the immune system, characterized by neutrophilia. CXC chemokines and CXC receptors are involved in this stress-induced redistribution of neutrophils from the hematopoietic tissue into the peripheral blood. This phenomenon is directly regulated by interactions between cortisol and the GR/MR. Considering the pivotal importance of neutrophilic granulocytes in the first line of defense, this knowledge is important for aquaculture, but will also contribute to the mechanisms involved in the stress-induced perturbation in neutrophil redistribution as often observed in clinical practice.

Extremely low frequency electromagnetic field exposure does not modulate toll-like receptor signaling in human peripheral blood mononuclear cells.

The effects of extremely low frequency electromagnetic fields (ELF-EMF) on human health remain unclear. It has been reported that ELF-EMF may modulate the innate immune response to microorganisms in animal models and mammalian cell-lines. With the recently gained insight in innate immune signaling and the discovery of pattern recognition, we aim to study whether ELF-EMF modulates innate inflammatory signaling pathways. We used human peripheral blood mononuclear cells (PBMCs), isolated from blood from healthy volunteers, which we stimulated with specific TLR2 and TLR4 ligands, and with several microorganisms. The cells were subsequently exposed in B(dc)=3 µT to a highly controlled and standardized ELF-EMF signal (20-5000Hz, B(ac)=5 µT, 30 min) and cytokine production was measured at different time points after stimulation. No significant difference in immune response, as reflected by IL-1ß, IL-6, TNFα, IL-8 and IL-10 production, could be detected after stimulation with LPS (TLR4 ligand), Pam3Cys (TLR2 ligand) or a panel of heat killed microorganisms: Mycobacterium tuberculosis, Salmonella typhimurium, Candida albicans, Aspergillus fumigatus and Staphylococcus aureus (multiple TLR ligands). We therefore conclude that under our experimental conditions, ELF-EMF does not modulate the innate immune response of human primary cells after TLR stimulation in vitro.

Morphine affects the inflammatory response in carp by impairment of leukocyte migration.

Opioid peptides are evolutionary conserved and in teleost fish their specific receptor types have been identified not only on neuroendocrine cells but also on immunocytes. In the present work we have studied the effects of morphine, ligand for the mu3 opioid receptor, on innate immune responses of common carp. Both in vitro and in vivo, during zymosan-induced peritonitis, morphine reduced gene expression of pro-inflammatory cytokines/chemokines and chemokine receptors. Furthermore, in vitro morphine administration also affects nitric oxide production, chemotaxis and apoptosis of head kidney leukocytes. These results provide evidence for an anti-inflammatory function of morphine and suggest an evolutionary conserved cross-talk between chemokines and opioids.

Cloning of opioid receptors in common carp (Cyprinus carpio L.) and their involvement in regulation of stress and immune response.

In mammals opiate alkaloids and endogenous opioid peptides exert their physiological and pharmacological actions through opioid receptors (MOR, DOR and KOR) expressed not only on neuroendocrine cells but also on leukocytes. Therefore, opioids can modulate the immune response. We cloned and sequenced all three classical opioid receptors (MOR, DOR and KOR) in common carp, and studied changes in their expression during stress and immune responses. Messenger RNA of opioid receptors was constitutively expressed in brain areas, specially in the preoptic nucleus NPO (homologous to mammalian hypothalamus). After exposure to prolonged restraint stress, mRNA levels of MOR and DOR decreased in the NPO and in the head kidney. Increased expression of all studied opioid receptors was observed in the pituitary pars distalis (containing ACTH-producing cells). In immune organs, constitutive but lower expression of opioid receptor genes was observed. During in vivo zymosan-induced peritonitis or after in vitro LPS-induced stimulation, when pro-inflammatory functions are activated, expression of the OR genes in leukocytes was concomitantly up-regulated. Additionally, specific agonists of opioid receptors especially reduced leukocyte migratory properties, manifested by reduced chemotaxis and down-regulated expression of chemokine receptors. Our data indicate an evolutionary conserved role for the opioid system in maintaining a dynamic equilibrium while coping with stress and/or infection.

The immune response differentially regulates Hsp70 and glucocorticoid receptor expression in vitro and in vivo in common carp (Cyprinus carpio L.).

Heat shock or stress proteins and glucocorticoids (cortisol) regulate a sequential pro-inflammatory and anti-inflammatory cytokine expression profile to effectively kill pathogens, whilst minimizing damage to the host. Cortisol elicits its effects through the glucocorticoid receptor (GR) for which Hsp70 and Hsp90 are required as chaperones. In common carp, (Cyprinus carpio) duplicated glucocorticoid receptor genes and splice variants with different cortisol sensitivities exist. We investigated the expression profiles of heat shock proteins Hsp70, Hsc70, Hsp90alpha and Hsp90beta and the three different variants of GR in vitro in and in vivo to define their role in immune modulation. A rapid transient induction of GR1 (a and b) and Hsp70 was seen after LPS treatment in vitro in head kidney phagocytes, whereas cortisol treatment did not affect constitutive or LPS-induced expression of Hsp70 or GR1 expression. In vivo zymosan-induced peritonitis upregulated GR and Hsp70 expression which appears to increase sensitivity for cortisol-induced immune modulation. Indeed, the increased GR and Hsp70 expression correlates with inhibition of both LPS- and zymosan-induced expression of pro-inflammatory cytokines. Infection with the blood parasite T. borreli decreases GR1a expression in thymus, but increases GR2 expression in spleen. Differentially regulated expression of Hsp70 and of glucocorticoid receptor variants with different cortisol sensitivities, underlines their physiological importance in a balanced immune response.

The presence of multiple and differentially regulated interleukin-12p40 genes in bony fishes signifies an expansion of the vertebrate heterodimeric cytokine family.

Interleukin-12 (IL-12) is the founding member of a rapidly growing family of heterodimeric cytokines. It consists of two subunits, designated p35 and p40 that together constitute a disulphide-linked heterodimeric cytokine. IL-12 is well known for its prominent role in both the early innate immune response and the skewing of the ensuing acquired immune response towards Th1. Here, we report the presence of IL-12p35 and three highly distinct IL-12p40 genes in common carp (Cyprinus carpio). The carp is a bony fish species genetically similar to the zebrafish, but its substantially larger body size facilitates immunological studies. A comparison of IL-12p35 genes of mammalian and non-mammalian species reveals the presence of a duplicated exon that is unique to the mammalian lineage. The organisation of the three carp IL-12p40 genes is similar to that of higher vertebrates. Phylogenetic analyses that include the p40-related subunits of other composite cytokines confirm the presence of three genuine IL-12p40 genes in carp and indicate that they are evolutionary ancient and possibly not restricted to bony fishes. The orthology of the different carp p40 subunits to mammalian IL-12p40 is further evident from the conservation of key residues involved in the formation of intra- and interchain disulphide bridges and the tight interlocking topology between p35 and p40. The expression of each of the carp IL-12p40 genes differs profoundly, constitutively as well as in response to in vitro stimulation of carp macrophages. Collectively, the presence of multiple and substantially different IL-12 genes signifies a considerable expansion of the vertebrate heterodimeric cytokine family.

Neuroendocrine-immune interaction: Evolutionarily conserved mechanisms that maintain allostasis in an ever-changing environment.

It has now become accepted that the immune system and neuroendocrine system form an integrated part of our physiology. Immunological defense mechanisms act in concert with physiological processes like growth and reproduction, energy intake and metabolism, as well as neuronal development. Not only are psychological and environmental stressors communicated to the immune system, but also, vice versa, the immune response and adaptation to a current pathogen challenge are communicated to the entire body, including the brain, to evoke adaptive responses (e.g., fever, sickness behavior) that ensure allocation of energy to fight the pathogen. This phenomenon is evolutionarily conserved. Hence it is both interesting and important to consider the evolutionary history of this bi-directional neuroendocrine-immune communication to reveal phylogenetically ancient or relatively recently acquired mechanisms. Indeed, such considerations have already disclosed an extensive "common vocabulary" of information pathways as well as molecules and their receptors used by both the neuroendocrine and immune systems. This review focuses on the principal mechanisms of bi-directional communication and the evidence for evolutionary conservation of the important physiological pathways involved.

The immunomodulatory role of the hypothalamus-pituitary-gonad axis: Proximate mechanism for reproduction-immune trade offs?

The present review discusses the communication between the hypothalamic-pituitary-gonad (HPG) axis and the immune system of vertebrates, attempting to situate the HPG-immune interaction into the context of life history trade-offs between reproductive and immune functions. More specifically, (i) we review molecular and cellular interactions between hormones of the HPG axis, and, as far as known, the involved mechanisms on immune functions, (ii) we evaluate whether the HPG-immune crosstalk serves as proximate mechanism mediating reproductive-immune trade-offs, and (iii) we ask whether the nature of the HPG-immune interaction is conserved throughout vertebrate evolution, despite the changes in immune functions, reproductive modes, and life histories. In all vertebrate classes studied so far, HPG hormones have immunomodulatory functions, and indications exist that they contribute to reproduction-immunity resource trade-offs, although the very limited information available for most non-mammalian vertebrates makes it difficult to judge how comparable or different the interactions are. There is good evidence that the HPG-immune crosstalk is part of the proximate mechanisms underlying the reproductive-immune trade-offs of vertebrates, but it is only one factor in a complex network of factors and processes. The fact that the HPG-immune interaction is flexible and can adapt to the functional and physiological requirements of specific life histories. Moreover, the assumption of a relatively fixed pattern of HPG influence on immune functions, with, for example, androgens always leading to immunosuppression and estrogens always being immunoprotective, is probably oversimplified, but the HPG-immune interaction can vary depending on the physiological and envoironmental context. Finally, the HPG-immune interaction is not only driven by resource trade-offs, but additional factors such as, for instance, the evolution of viviparity shape this neuroendocrine-immune relationship.

A role for multiple estrogen receptors in immune regulation of common carp.

Estrogens are important for bi-directional neuroendocrine-immune interaction. They act via nuclear estrogen receptors (ERα and ERß) and/or G-protein coupled receptor - GPR30. We found expression of ERα, ERß and GPR30 in carp lymphoid tissues and head kidney monocytes/macrophages, neutrophils and lymphocytes. Interestingly, ERß is also expressed in some head kidney lymphocytes but not in naive PBLs. Immune stimulation altered the cell type specific profile of expression of these receptors, which depends on both activation and maturation stage. This implies direct leukocyte responsiveness to estrogen stimulation and therefore in vitro effects of 17ß-estradiol (E2) on reactive oxygen species (ROS) production in monocytes/macrophages were determined. Short-time incubation with E2 increased ROS production in PMA-stimulated cells. Results comply with mediation by GPR30, partially functioning via phosphoinositide 3-kinase activation. These results furthermore demonstrate that neuroendocrine-immune communication via estrogen receptors is evolutionary conserved.

Increased leptin expression in common Carp (Cyprinus carpio) after food intake but not after fasting or feeding to satiation.

Leptin is a key factor in the regulation of food intake and is an important factor in the pathophysiology of obesity. However, more than a decade after the discovery of leptin in mouse, information regarding leptin in any nonmammalian species is still scant. We report the identification of duplicate leptin genes in common carp (Cyprinus carpio). The unique gene structure, the conservation of both cysteines that form leptin's single disulfide bridge, and stable clustering in phylogenetic analyses substantiate the unambiguous orthology of mammalian and carp leptins, despite low amino acid identity. The liver is a major yet not the only site of leptin expression. However, neither 6 d nor 6 wk of fasting nor subsequent refeeding affected hepatic leptin expression, although the carp predictably shifted from carbohydrate to lipid metabolism. Animals that were fed to satiation grew twice as fast as controls; however, they did not show increased leptin expression at the termination of the study. Hepatic leptin expression did, however, display an acute and transient postprandial increase that follows the postprandial plasma glucose peak. In summary, leptin mRNA expression in carp changes acutely after food intake, but involvement of leptin in the long-term regulation of food intake and energy metabolism was not evident from fasting for days or weeks or long-term feeding to satiation. These are the first data on the regulation of leptin expression in any nonmammalian species.

Central and peripheral interleukin-1beta and interleukin-1 receptor I expression and their role in the acute stress response of common carp, Cyprinus carpio L.

In fish, the hypothalamus-pituitary-interrenal axis (HPI-axis), the equivalent of the hypothalamus-pituitary-adrenal axis (HPA-axis) in mammals, is activated during stress and leads to production and release of cortisol by the interregnal cells in the head kidney. In mammals, the cytokine interleukin-1beta (IL-1beta) takes a key position in the innate immune and inflammatory responses and influences the HPA-axis. In fish, studies that address the effects of cytokines on HPI-axis activation are limited. We quantitatively assessed expression of IL-1beta and its receptor, IL-1RI (the latter was cloned and sequenced), in an acute restraint stress paradigm in common carp, Cyprinus carpio. We also considered expression of the pituitary hormones prolactin (PRL) and GH that have been shown to be structurally related to cytokines and have immunomodulatory actions. Pituitary PRL expression increased fourfold during stress; GH mRNA levels were unaffected. Following restraint, hypothalamic IL-1beta expression was upregulated; in head kidney and pituitary pars intermedia, IL-1RI expression significantly increased. We suggest that during acute stress IL-1beta signalling in the HPI-axis becomes more sensitive, since both ligand and receptor expressions are enhanced. In vitro, recombinant carp IL-1beta stimulates release of alpha-MSH and N-Ac beta-endorphin from the pituitary gland. This observation concurs with increased in vivo plasma levels of alpha-MSH and N-Ac beta-endorphin following restraint. Our findings combined lead us to conclude that IL-1beta affects the activity of the HPI-axis and, in turn, expression profiles of genes encoding IL-1beta and its receptor are modified during acute stress. Our study provides convincing evidence for bi-directional communication of the HPI-axis and the immune system in fish.