Article

Modulatory in vitro effect of stress hormones on the cytokine response of rainbow trout and gilthead sea bream head kidney stimulated with Vibrio anguillarum bacterin

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Abstract

In fish, the stress response and their consequences in the immune system have been widely described. Recently, a differential cytokine regulation between rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) was reported after treatment with stress hormones together with their receptor antagonists. Nevertheless, there is no evidence of whether antagonists for stress hormone receptors may influence the interaction between hormones and cytokines after bacterial administration. Thus, the aim of our study was to evaluate the cytokine expression in the presence of stress hormones (cortisol, ACTH, adrenaline), hormone receptor antagonists and inactivated Vibrio anguillarum bacterin in rainbow trout and gilthead sea bream head kidney primary cell culture (HKPCC). Mifepristone, spironolactone, propranolol and phentolamine were used to block GR, MR, MC2R, and β-/α-adrenoreceptors. Our results showed an expected increase of the pro-inflammatory and anti-inflammatory response after inactivated V. anguillarum bacterin treatment in both species. Cortisol, ACTH and adrenaline did not modulate the expression of immune-related genes in rainbow trout, while in sea bream cortisol was able to reduce the stimulated gene expression of all cytokines. This effect was only restored to basal expression level in IL-1β and TNF-α by mifepristone. ACTH reduced both pro-inflammatory and anti-inflammatory cytokine expression, excluding IL-1β, only in sea bream. Adrenaline enhanced the expression of IL-1β and TGF-β1 stimulated by inactivated V. anguillarum in sea bream, and the effect was diminished by propranolol. In sum, our results confirm that the immunoendocrine differences reported at gene expression profile between two teleost species are also observed after exposure to inactivated V. anguillarum bacterin, suggesting that stress hormones would differentially modulate the immune response against pathogens in teleost species.

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... In response to stressful conditions, cortisol increases the metabolic capacity of the liver to meet the increased energy demand [336]. In addition, cortisol has been reported to reduce the number of circulating lymphocytes [338] and restrict the expression of distinct immune genes in teleostean immune cells, for instance, pro-inflammatory interleukin genes and/or acute-phase genes, in part together with the simultaneous upregulation of socs1 and -2 [329,[339][340][341]. The effects of cortisol are mediated by the glucocorticoid receptor (GR). ...
... Similar with cortisol, ACTH has a comparably profound impact on innate immunity. Stimulation with ACTH increased the transcript levels of the anti-inflammatory mediators tgfb1, il6, il10 and also of pro-inflammatory tnf in head-kidney cells from sea bream [340,341] although it failed to modulate the cytokine expression in head-kidney cells from rainbow trout [340]. ...
... Similar with cortisol, ACTH has a comparably profound impact on innate immunity. Stimulation with ACTH increased the transcript levels of the anti-inflammatory mediators tgfb1, il6, il10 and also of pro-inflammatory tnf in head-kidney cells from sea bream [340,341] although it failed to modulate the cytokine expression in head-kidney cells from rainbow trout [340]. ...
Article
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The innate immune response involves a concerted network of induced gene products, preformed immune effectors, biochemical signalling cascades and specialised cells. However, the multifaceted activation of these defensive measures can derail or overshoot and, if left unchecked, overwhelm the host. A plenty of regulatory devices therefore mediate the fragile equilibrium between pathogen defence and pathophysiological manifestations. Over the past decade in particular, an almost complete set of teleostean sequences orthologous to mammalian immunoregulatory factors has been identified in various fish species, which prove the remarkable conservation of innate immune-control concepts among vertebrates. This review will present the current knowledge on more than 50 teleostean regulatory factors (plus additional fish-specific paralogs) that are of paramount importance for controlling the clotting cascade, the complement system, pattern-recognition pathways and cytokine signalling networks. A special focus lies on those immunoregulatory features that have emerged as potential biomarker genes in transcriptome-wide research studies. Moreover, we report on the latest progress in elucidating control elements that act directly with immune-gene-encoding nucleic acids, such as transcription factors, hormone receptors and micro- and long noncoding RNAs. Investigations into the function of teleostean inhibitory factors are still mainly based on gene-expression profiling or overexpression studies. However, in support of structural and in-vitro analyses, evidence from in-vivo trials is also available and revealed many biochemical details on piscine immune regulation. The presence of multiple gene copies in fish adds a degree of complexity, as it is so far hardly understood if they might play distinct roles during inflammation. The present review addresses this and other open questions that should be tackled by fish immunologists in future.
... In response to stressful conditions, cortisol increases the metabolic capacity of the liver to meet the increased energy demand [336]. In addition, cortisol has been reported to reduce the number of circulating lymphocytes [338] and restrict the expression of distinct immune genes in teleostean immune cells, for instance, pro-inflammatory interleukin genes and/or acute-phase genes, in part together with the simultaneous upregulation of socs1 and -2 [329,[339][340][341]. The effects of cortisol are mediated by the glucocorticoid receptor (GR). ...
... Similar with cortisol, ACTH has a comparably profound impact on innate immunity. Stimulation with ACTH increased the transcript levels of the anti-inflammatory mediators tgfb1, il6, il10 and also of pro-inflammatory tnf in head-kidney cells from sea bream [340,341] although it failed to modulate the cytokine expression in head-kidney cells from rainbow trout [340]. ...
... Similar with cortisol, ACTH has a comparably profound impact on innate immunity. Stimulation with ACTH increased the transcript levels of the anti-inflammatory mediators tgfb1, il6, il10 and also of pro-inflammatory tnf in head-kidney cells from sea bream [340,341] although it failed to modulate the cytokine expression in head-kidney cells from rainbow trout [340]. ...
... Cortisol and its receptors [glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) (9)] play an important role in regulating crosstalk between the stress response and immune networks. Activation of the GR (or MR) may serve as an early danger alarm and enable the immune system to prepare for the fight against health challenges (10,11). Moreover, GR (or MR) activation modulates the leukocyte-regulated immune responses and negotiates the initiation and efficacy of immune functions (1). ...
... In response to pathogen infection, the homeostatic interaction between the stress response and cytokine-induced inflammation in teleosts is more complicated, showing no negative or positive correlation among various teleosts. For example, the stress response (mimicked by cortisol) does not affect cytokine gene expression in rainbow trout (Oncorhynchus mykiss); however, the stress response did reduce the stimulated gene expression of all cytokines in gilthead sea bream (Sparus aurata) (11). In the European sea bass (Dicentrarchus labrax), genes associated with glucocorticoid synthesis and inflammatory responses are simultaneously upregulated after Vibrio anguillarum infection (5). ...
... Infectious diseases are constant threats to aquaculture and larviculture, causing significant financial losses due to high infectivity and mortality (11). V. anguillarum, the causative agent of vibriosis, is a gram-negative bacteria that causes severe, frequently deadly hemorrhagic septicemia in teleosts (26,27). ...
Article
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Rainbow trout ( Oncorhynchus mykiss ) is one of the most common aquaculture fish species worldwide. Vibriosis disease outbreaks cause significant setbacks to aquaculture. The stress and immune responses are bidirectionally modulated in response to the health challenges. Therefore, an investigation into the regulatory mechanisms of the stress and immune responses in trout is invaluable for identifying potential vibriosis treatments. We investigated the transcriptional profiles of genes associated with stress and trout immune functions after Vibrio anguillarum infection. We compared the control trout (CT, 0.9% saline injection), asymptomatic trout (AT, surviving trout with minor or no symptoms after bacteria injection), and symptomatic trout (ST, moribund trout with severe symptoms after bacteria injection). Our results showed activated immunomodulatory genes in the cytokine network and downregulated glucocorticoid and mineralocorticoid receptors in both AT and ST, indicating activation of the proinflammatory cytokine cascade as a common response in AT and ST. Moreover, the AT specifically activated the complement- and TNF-associated immune defenses in response to V. anguillarum infection. However, the complement and coagulation cascades, as well as steroid hormone homeostasis in ST, were disturbed by V. anguillarum . Our studies provide new insights toward understanding regulatory mechanisms in stress and immune functions in response to diseases.
... Like other members of the family, GR possesses a modular structure consisting of three major domains: the N-terminal activation function-1 domain (AF-1), DNA binding domain (DBD), and a C-terminal ligand binding domain (LBD) [10]. GR signaling interacts with the immune system through two different mechanisms, namely genomic and non-genomic [11]. Upon ligand binding, the GR translocates into the nucleus, where it can regulate the expression of a diverse range of inflammatory and anti-inflammatory genes [12]. ...
... GR limits mortality and cytokine production by inducing anti-inflammatory genes [15] and protects macrophages in an LPS-induced shock model [14]. In monocytes, GR signaling is involved in the regulation of apoptosis, adhesion, chemotaxis, phagocytosis, and reactive oxygen metabolism, and can influence monocyte targeting to specific macrophage subpopulations [7,11]. In mice, it has been found that GR signaling in macrophages is involved in cell-and tissue-specific actions of glucocorticoids and plays a crucial role in tissue-repair mechanisms [16]. ...
... In rainbow trout, Co-stimulation of cortisol with the inflammatory agents resulted in the upregulated of IL-10, and the down-regulated of IL-6 and IL-8 in macrophage cell line [7]. Furthermore, cortisol does not show significant modulatory effects on cytokine expression induced by V. anguillarum bacteria in the rainbow trout macrophage cell line, while in sea bream cortisol did produce a clear inhibitory effect on both pro-inflammatory and anti-inflammatory cytokines by V. anguillarum bacterial infection in head kidney cells [11]. These results indicate that there are species differences in GR activity during immune regulation. ...
Article
The glucocorticoid receptor (GR) is an important feedback regulator of the hypothalamic-pituitary-interrenal (HPI) axis. However, there are a limited number of studies focused on host-pathogen interactions in which an association between GR and immune response has been evaluated in monocytes/macrophages (MO/MФ) after being challenged with highly pathogenic bacteria. Here, we cloned the cDNA sequence of the glucocorticoid receptor (PaGR) gene from ayu fish. The PaGR transcript was expressed in all tissues, and changes in expression were observed in immune tissues and MO/MФ after live Vibrio anguillarum infection. Subsequently, PaGR was expressed and purified to prepare anti-PaGR antibodies. We analyzed the subcellular localization of PaGR. PaGR was expressed not only in the intracellular space but also in the plasma membrane. PaGR activation decreased the expression of pro-inflammatory cytokines and increased the expression of anti-inflammatory cytokines. However, PaGR activation suppressed the phagocytosis activity of V. anguillarum-infected ayu MO/MФ via a non-genomic pathway. Interestingly, PaGR activation could enhance MO/MФ bacterial killing capability and apoptosis. Therefore, PaGR may modulate the immune response in ayu MO/MФ by genomic and non-genomic pathways.
... In fish, these catecholamines bind to α-and β-adrenoreceptors (ADRA, ADRB) in the membrane and modulate, together with other hormones, a multitude of physiological processes [14,15]. In particular, adrenaline and noradrenaline inhibit antibody production and suppress pro-inflammatory cytokines and phagocytic activity [16,17]. However, human monocytes showed enhanced pro-inflammatory responses when stimulated with an ADRB agonist [18]. ...
... Altogether, the effects of stress on the teleostean immune system are highly complex, since they depend on the type of stressor and its exposure time as well as the identity of the affected (immune-) cell populations and the species. So far, the influence of stress hormones in general, and catecholamines in particular, on the expression of immune genes in head-kidney cells has rarely been examined and provided in part contradictory results [16,17,27]. ...
... Cortisol-immune interactions have been well explored in studies on primary [13,16,17,79,80] and secondary [81] cell cultures from various fish species, most of which agree that cortisol suppresses the stimulation-dependent synthesis of cytokines, SAA and other pro-inflammatory mediators. Although considered anti-inflammatory, cortisol on its own initially (after 3 h) upregulated the pro-inflammatory cytokine genes IL1B, IL6, and TNF and downregulated them after 24 h of incubation. ...
Article
Full-text available
The functional spectrum of the teleostean head kidney covers haematopoietic, immune and endocrine signalling pathways with physiological effects that are likely to conflict if activated at the same time. An in vivo experiment on the salmonid fish maraena whitefish (Coregonus maraena) revealed that the head kidney shows a remarkably strong response after injection of Aeromonas salmonicida within 48 h. In order to investigate the potential influence of endocrine signalling on the initiation of immune responses, we established a primary head-kidney cell culture of maraena whitefish. For the characterisation of this model system, we used flow cytometry complemented with an extensive panel of immunological/haematological and stress-physiological/neuroendocrinological qPCR assays. More than half of the cells expressed the characteristic signature of myeloid cells, while more than one third of the cells expressed those genes typical for lymphocytes and monocytes. In parallel, we quantified the expression of genes encoding endocrine receptors and identified ADRA2D as by far the most highly expressed adrenergic-receptor gene in head-kidney cells. The stimulation of the primary head-kidney cells with toll-like receptor ligands induced the expression of typical immune genes (IL1B, CXCL8, TNF, SAA) after only 1 h. The incubation with the stress hormones cortisol, adrenaline and noradrenaline also had an immune-activating effect, though less pronounced. However, cortisol had the strongest suppressive effect on the stimulation-induced immune response, while adrenaline exerted a comparably weaker effect and noradrenaline was almost ineffective. Moreover, we found that cortisol reduced the expression of genes coding for adrenergic and some glucocorticoid receptors, while noradrenaline increased it. In conclusion, the primary head-kidney cells of maraena whitefish reflect the immunological and neuroendocrinological diversity of the entire organ. This in vitro system allowed thus identifying the correlative changes between the activities of hormones and immune factors in salmonid fish in order to contribute to a better understanding of the regulation circuit between stress and immune defence.
... It has been extensively described that stress and immune response are tightly connected (35,36). Many studies have reported that a stressor induces alterations on innate immune response (37)(38)(39). ...
... In sea bream, cortisol showed the ability to decrease the expression of pro-inflammatory cytokines both in leukocytes from the head kidney (42) and also in head kidney primary cell culture (HKPCC) (40). The same immunosuppresor cortisol effect was observed in sea bream HKPCC stimulated with inactivated V. anguillarum (35). This evidence, in combination with the results reported in our study, suggests that the high level of cortisol observed at 120 hpc in sea bass larvae challenged with V. anguillarum is a response mechanism attributed to the sensing of a biological threat related to the augmented pro-inflammatory response registered at 120 hpc. ...
Article
Although several efforts have been made to describe the immunoendocrine interaction in fish, there are no studies to date focusing on the characterization of the immune response and glucocorticoid synthesis using the host–pathogen interaction on larval stage as an early developmental stage model of study. Therefore, the aim of this study was to evaluate the glucocorticoid synthesis and the modulation of stress- and innate immune-related genes in European sea bass (Dicentrarchus labrax) larvae challenged with Vibrio anguillarum. For this purpose, we challenged by bath full-sibling gnotobiotic sea bass larvae with 107 CFU mL−1 of V. anguillarum strain HI 610 on day 5 post-hatching (dph). The mortality was monitored up to the end of the experiment [120 hours post-challenge (hpc)]. While no variations were registered in non-challenged larvae maintained under gnotobiotic conditions (93.20% survival at 120 hpc), in the challenged group a constant and sustained mortality was observed from 36 hpc onward, dropping to 18.31% survival at 120 hpc. Glucocorticoid quantification and expression analysis of stress- and innate immunity-related genes were carried out in single larvae. The increase of cortisol, cortisone and 20β-dihydrocortisone was observed at 120 hpc, although did not influence upon the modulation of stress-related genes (glucocorticoid receptor 1 [gr1], gr2, and heat shock protein 70 [hsp70]). On the other hand, the expression of lysozyme, transferrin, and il-10 differentially increased at 120 hpc together with a marked upregulation of the pro-inflammatory cytokines (il-1β and il-8) and hepcidin, suggesting a late activation of defense mechanisms against V. anguillarum. Importantly, this response coincided with the lowest survival observed in challenged groups. Therefore, the increase in markers associated with glucocorticoid synthesis together with the upregulation of genes associated with the anti-inflammatory response suggests that in larvae infected with V. anguillarum a pro-inflammatory response at systemic level takes place, which then leads to the participation of other physiological mechanisms at systemic level to counteract the effect and the consequences of such response. However, this late systemic response could be related to the previous high mortality observed in sea bass larvae challenged with V. anguillarum.
... An exception was observed in tnfα mRNA levels in fish exposed to 0.02 mg/L PSNPs which presented an up-regulation. Based upon well stablished studies of pathogenchallenged fish, il1β is considered a canonical pro-inflammatory cytokine, released at the early stages of inflammatory responses, activating phagocytic and lymphocytic populations, cytokine cascades, and acting as a upstream regulator of innate and adaptive defensive responses [72,73]. IL8 also contributes to the unfolding of inflammatory responses by acting as a chemoattractant for neutrophils and other early stage immune cells to the site of infection, enhancing the acute phase response upon bacterial or viral infection [74]. ...
... Nevertheless, up to date, only a few studies have evaluated the effects of exposure to nanoparticles (gold [88], silver [89] and plastic [19] nanoparticles) on skin mucus. During a short-term pathogen-mediated inflammation or stress-related activation of immune responses in fish, both plasma and skin cortisol show an species-and stressor-specific pattern that often follows a peak of secretion at 6-12h, that may suppress immune response or, especially in short-term stress, activate mucosal immunity [39,73,90]. Low levels of glucose have been described to trigger cortisol secretion in fish exposed to PSNP [91], but the present data show no changes in glucose levels at 96h. ...
Article
Pernicious effects of plastic particles, emergent contaminants worldwide, have been described in different species. In teleost species, alterations of immune function after exposure to nanoplastics (NPs) have been reported, but the interaction with cortisol – hypothalamic–pituitary–adrenal (HPI) axis has not yet been explored. Furthermore, the role of dissolved organic matter on the effects of NPs is poorly known. Thus, the aims of this research were to assess if polystyrene NPs (PSNPs) acted as a stressor on juvenile European seabass (Dicentrarchus labrax), interfering with the immune response, as well as to elucidate if humic acids (HA) modulated the potential effects of PSNPs. A short-term exposure to PSNPs and HA elicited an immuno-modulatory response, with an activation of steroidogenic stress-related pathways. An upregulation of anti-inflammatory cytokine (il10, tgfb) and stress-related (mc2r, gr1) transcripts were observed after exposure to HA and PSNPs both individually and in co-exposure. No notable alteration of inflammatory markers was consistently found, which may reflect a protective anti-inflammatory effect of HA in the presence of PSNPs. Nevertheless, there seems to be a more complex interaction between both components. Overall, data show that understanding the interaction of NPs with dissolved organic substances is key to deciphering their environmental risks.
... Therefore, it is necessary to evaluate the impact of the combination of stressors on the activation of the HPI axis and the expression of cytokines in different organs implicated in the immune and stress response in different teleost species. We recently revealed that, although most fish show a generalized stress reaction in vitro and in mucosae, the pattern and magnitude of the response may be affected not only by environmental factors (such as temperature and salinity) but also by the nature of stressors and distinct evolutionary life stories [8,9]. Therefore, an experimental ground for the description of common and specific stress dynamics may require a comparative approach per species to short-term responsiveness to common aquaculture stressors. ...
... Interestingly, the expression of pro-inflammatory il1β in liver and spleen of stressed or vaccinated seabream increased up to 8-fold but only up to 2-fold in zebrafish and trout (Fig. 5). This illustrates a species-specific pattern of short-term gene activation for il1β that has been described previously [9,24]. ...
Article
The stress and immune-related effects of short-term (1, 6 and 24 h) air exposure stress (1 min), bath vaccination with Vibrio anguillarum bacterin, and both stressors combined were evaluated in liver and spleen of Sparus aurata, Danio rerio and Onchorhynchus mykiss. Expression profiles of immune (interleukin 1 beta: il1β; tumor necrosis factor alpha: tnfα; interleukin 10: il10; tumor growth factor beta: tgfβ1; immunoglobulin M: igm; lysozyme: lys; complement protein c3: c3) and stress-related genes (glucocorticoid receptor: gr; heat shock protein 70: hsp70; and enolase) were analysed by RT-qPCR. Cortisol level was assessed by radioimmunoassay. The gene expression patterns in liver and spleen were found to be differentially regulated in a time- and organ-dependent manner among species. In seabream, a higher il1β-driven inflammatory response was recorded. In zebrafish, air exposure stress but not bath vaccination alone modulated most of the changes in liver and spleen immune transcripts. Stressed and vaccinated trout showed an intermediate pattern of gene expression, with a lower upregulation of immune-related genes in liver and the absence of changes in the expression of hsp70 and enolase in spleen (as it was observed in seabream but not in zebrafish). Following air exposure, cortisol levels increased in plasma 1 h post-stress (hps) and then decreased at 6 hps in O. mykiss and D. rerio. By contrast, in S.aurata the cortisol level remained higher at 6 hps suggesting a greater degree of responsiveness to this stressor. When fish were exposed to combined air exposure plus bath vaccination cortisol levels were also augmented at 1 and 6 hps in O. mykiss and S.aurata and restored to basal level at 24 hps, whereas in D. rerio the response was higher in response to the combination of both stressors. In addition, V. anguillarum bacterin vaccination triggered cortisol secretion only in D. rerio, suggesting a greater responsiveness of D. rerio hypothalamic-pituitary-interrenal axis. Overall, comparing the tissue transcription responsiveness, liver was found to be more implicated in the response to handling stress compared to spleen. These results also indicate that a species-specific response accounts for the deviations of stress and immune onset in the liver and spleen in these fish species.
... The immune-privileged tissue concept suggests the existence of different conditions that help to control the access of pathogens to the central nervous system (CNS) but also the exacerbation of inflammation [31]. This is why both pro-inflammatory and anti-inflammatory cytokines are overexpressed after stimulation by several types of stressors, including pathogens and vaccines [32]. These responses are thought to be an evolutionary adaptation to protect indispensable organs with limited regeneration capacities from uncontrolled inflammation [33]. ...
... Indeed, the downregulation of pituitary hormones could be explained by the negative feedback established between cortisol release and the recurrent synthesis of hypothalamic and pituitary hormones [35]. Similar results were observed in the rainbow trout head kidney after treatment with Vibrio bacterin [32]. Moreover, our results are supported by a recent paper on the effects of vaccine exposure in seabream (Sparus aurata). ...
Article
Full-text available
Nodavirus, or nervous necrosis virus (NNV ), is the causative agent of viral encephalopathy and retinopathy (VER), a severe disease affecting numerous fish species worldwide. European sea bass, a cultured species of great economic importance, is highly susceptible to the disease. To better understand the response of this organism to NNV, we conducted RNA‑Seq analysis of the brain and head kidney from experimentally infected and uninfected sea bass juveniles at 24 and 72 hours post‑infection (hpi). Contrary to what was expected, we observed modest modulation of immune‑related genes in the brain, the target organ of this virus, and some of these genes were even downregulated. However, genes involved in the stress response showed extremely high modulation. Accordingly, the genes encoding the enzymes implicated in the synthesis of cortisol were almost the only overexpressed genes in the head kidney at 24 hpi. This stress response was attenuated after 72 h in both tissues, and a progressive immune response against the virus was mounted. Moreover, experiments were conducted to determine how stress activation could impact NNV replication. Our results show the complex interplay between viral activity, the stress reaction and the immune response.
... An exception was observed in tnfα mRNA levels in fish exposed to 0.02 mg/L PSNPs which presented an up-regulation. Based upon well stabilished studies of pathogen-challenged fish, il1β is considered a canonical pro-inflammatory cytokine, released at the early stages of inflammatory responses, activating phagocytic and lymphocytic populations, cytokine cascades, and acting as a upstream regulator of innate and adaptive defensive responses (Zhu et al., 2013;Khansari et al., 2017). IL8 also contributes to the unfolding of inflammatory responses by acting as a chemoattractant for neutrophils and other early stage immune cells to the site of infection, enhancing the acute phase response upon bacterial or viral infection (Moreno et al., 2018). ...
... Nevertheless, up to date, only a few studies have evaluated the effects of exposure to nanoparticles (gold , silver (Hawkins et al., 2014) and plastic (Brandts et al., 2018) nanoparticles) on skin mucus. During a short-term pathogen-mediated inflammation or stress-related activation of immune responses in fish, both plasma and skin cortisol show a species-and stressor-specific pattern that often follows a peak of secretion at 6-12 h, that may suppress immune response or, especially in short-term stress, activate mucosal immunity (Lieke et al., 2021;Khansari et al., 2017Khansari et al., , 2018. Low levels of glucose have been described to trigger cortisol secretion in fish exposed to PSNP (Brun et al., 2019), but the present data show no changes in glucose levels at 96 h. ...
Chapter
Small plastic particles are considered emerging pollutants, and this has motivated research to establish their ecological and environmental consequences. Currently, the study of the effects of nanoplastics (NPs) in aquatic organisms is still scarce, especially in organisms of higher trophic levels, such as fish.
... Moreover, little attention has been paid on the interaction and cross-modulatory effects between endocrine and immune systems among different fish species under stress situations. In fact, it has been recently reported that the combination of stress hormones and pathogen antigens could differentially induce a species-specific response (29). On the other hand, at the local response level, few studies have addressed the effects of stressful stimuli on the fish mucosal immune system. ...
Article
Fish have to face various environmental challenges that may compromise the efficacy of the immune response in mucosal surfaces. Since the effect of acute stress on mucosal barriers in fish has still not been fully elucidated, we aimed to compare the short-term mucosal stress and immune transcriptomic responses in a freshwater (rainbow trout, Oncorhynchus mykiss) and a marine fish (gilthead seabream, Sparus aurata) to bacterial immersion (Vibrio anguillarum bacterin vaccine) and air exposure stress in skin, gills, and intestine. Air exposure and combined (vaccine + air) stressors exposure were found to be inducers of the cortisol secretion in plasma and skin mucus on both species in a time-dependent manner, while V. anguillarum bacterin exposure induced cortisol release in trout skin mucus only. This was coincident with a marked differential increase in transcriptomic patterns of stress-and immune-related gene expression profiles. Particularly in seabream skin, the expression of cytokines was markedly enhanced, whereas in gills the response was mainly suppressed. In rainbow trout gut, both air exposure and vaccine stimulated the transcriptomic response, whereas in seabream, stress and immune responses were mainly induced by air exposure. Therefore, our comparative survey on the transcriptomic mucosal responses demonstrates that skin and gut were generally more reactive in both species. However, the upregulation of immune transcripts was more pronounced in gills and gut of vaccinated trout, whereas seabream appeared to be more stress-prone and less responsive to V. anguillarum bacterin in gills and gut. When fish were subjected to both treatments no definite pattern was observed. Overall, the results indicate that (1) the immune response was not homogeneous among mucosae (2), it was greatly influenced by the specific traits of each stressor in each surface and (3) was highly species-specific, probably as a result of the adaptive life story of each species to the microbial load and environmental characteristics of their respective natural habitats.
... They are also the major source for the secretion of pro-inflammatory or anti-inflammatory cytokines under pathological conditions [17,45]. Cytokines such as IL-1β, IL-10, TGF-β, and TNF-α, play key roles in mediating acute inflammatory reactions [46][47][48]. According to previous reports, fADP induces the production of anti-inflammatory mediators IL-10 in human macrophages, and significantly impairs the production of the pro-inflammatory cytokine such as TNF-α [49]. ...
Article
Adiponectin (ADP), a regulator of the innate immune system, plays a role in the progression of inflammation and metabolic disorders in mammals. However, the role of ADP in fish is poorly understood. Here, we cloned the cDNA sequence of a ADP homolog (PaADP) gene from ayu. Multiple sequence alignment revealed that PaADP presented typical characteristics of ADPs. Phylogenetic tree analysis showed that PaADP was most closely related to that of rainbow trout. In healthy ayu, the transcripts of PaADP were detected in most of the tested tissues and cells, with the highest level in the adipose tissue. Upon V. anguillarum infection, the mRNA expression of PaADP was significantly up-regulated in the tissues and cells except adipose tissue. Subsequently, the full-length mature PaADP (fPaADP) and the globular domain fragment (gPaADP) were prokaryotically expressed in bacteria and purified, and anti-PaADP antibodies were produced. Western blot analysis revealed that three fragments including fPaADP and gPaADP were existed in ayu serum. The recombinant fPaADP (rfPaADP) had an anti-inflammatory effect on ayu MO/MФ by upregulating anti-inflammatory cytokine expressions, downregulating pro-inflammatory cytokine expressions, inhibiting the phagocytosis and subsequent bacterial killing. In contrast, the recombinant gPaADP (rgPaADP) presented a pro-inflammatory effect on ayu MO/MФ by upregulating pro-inflammatory cytokine expression, downregulating anti-inflammatory cytokine expressions, enhancing the phagocytosis and subsequent bacterial killing. These results suggested that fPaADP and gPaADP have opposite roles in the regulation of MO/MФ functions in ayu.
... After placing fish into the keepnets, a significant rise in PCC was detected, and circulating levels remained high up to 24 h after the start of the experiment. In response to an acute stressor, circulating cortisol levels in rainbow trout peak within 10 min to 1 h and return to basal within 8 h (De Mercado et al., 2018;Gesto et al., 2013;Khansari et al., 2018;Khansari et al., 2017;López-Patiño et al., 2014). While the later studies reported an increase of 2 to 4 times in blood cortisol values in response to acute stressors, in our study peak cortisol levels were about 11 times higher than pre-stress levels, probably given by the severity and the duration of the continuous stressor applied (Barton, 2002). ...
Article
Cortisol, the end product of the hypothalamus-pituitary-interrenal (HPI) axis, has been traditionally measured in blood as indicator of stress in fish, however, the degree of invasiveness inherent to blood collection is not always possible or desirable. Instead, cortisol measurement in skin mucus is far less invasive, but as blood, this method potentially provides only a brief window of information of the HPI axis activity. The newly described method of cortisol measurement from scales may serve as a long-term, integrated measure of the HPI axis activity in fish. While skin mucus and scales cortisol measurement present practical and conceptual advantages, there are still several unclear issues related to their biological relevance that need deeper study. Accordingly, we aimed to evaluate whether skin mucus and scales cortisol levels can be reliably used as stress indicators by subjecting fish to prolonged, continuous stressful conditions. The present study demonstrates that the measurement of cortisol in skin mucus reflect circulating cortisol concentrations when fish are responding to stress with an intense activation of the HPI axis. Results also revealed that cortisol content in scales strongly correlates to circulating cortisol levels in chronically stressed fish. Besides, we provide further support that scales cortisol assessment offer a retrospective measure of the past stress experience in fish. While this study provides a good basis for future research applying the methods presented, our results open the question of whether these matrices have additional sources of cortisol other than blood, and the route of incorporation or diffusion. Further knowledge about the general robustness and stability of scales cortisol in fish subjected to prolonged stress would largely help strengthen the interpretation of hormone fluctuations in this matrix.
... Vaccination is the most effective method used nowadays in aquaculture to prevent diseases caused by pathogens (Plant and LaPatra, 2011). Available data indicates that 2 h after Vibrio anguillarum bacterin exposure, the expression of both pro-and anti-inflammatory genes increase in gilthead seabream (Sparus aurata) head kidney primary cell culture (Khansari et al., 2017). Moreover, vaccination by immersion leads to alteration of some immune genes including complement c3, tumor necrosis factor alpha (tnfα), lysozyme (lys) or transforming-growth factor beta (tgfβ) in seabream mucosal tissues such as skin and gut (Khansari et al., 2018). ...
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Vaccination is a widely used therapeutical strategy in aquaculture, but whether vaccination elicits stress responses in the central neuroendocrine system and enhances the crosstalk between the immune and endocrine systems in the brain or pituitary after vaccination is unclear. To answer this question two experiments using two different vaccine exposure routes, i.e., bath or intraperitoneal (i.p.) injection, were carried out on gilthead seabream (Sparus aurata L.). In the first one, the stress responses of fish subjected to waterborne Vibrio anguillarum bacterin were compared with responses after air exposure or their combination. In the second experiment, fish were subjected to an intraperitoneal injection of Lactococcus garvieae bacterin and we assessed the central stress response and also whether or not a significant immune response was induced in brain and pituitary. In both experiments, blood, brain and pituitary tissues were collected at 1, 6, and 24 h post stress for plasma hormone determination and gene expression analysis, respectively. Results indicated that bath vaccination induced a decreased central stress response compared to air exposure which stimulated both brain and pituitary stress genes. In the second experiment, injection vaccination kept unchanged plasma stress hormones except cortisol that raised at 6 and 24 h. In agreement, non-significant or slight changes on the transcription of stress-related genes were recorded, including the hormone genes of the hypothalamic pituitary interrenal (HPI) axis and other stress markers such as hsp70, hsp90, and mt genes in either brain or pituitary. Significant changes were observed, however, in crhbp and gr. In this second experiment the immune genes il1β, cox2, and lys, showed a strong expression in both brain and pituitary after vaccination, notably il1β which showed more than 10 fold raise. Overall, vaccination procedures, although showing a cortisol response, did not induce other major stress response in brain or pituitary, regardless the administration route. Other than main changes, the alteration of crhbp and gr suggests that these genes could play a relevant role in the feedback regulation of HPI axis after vaccination. In addition, from the results obtained in this work, it is also demonstrated that the immune system maintains a high activity in both brain and pituitary after vaccine injection.
... Vaccination is the most effective method used nowadays in aquaculture to prevent diseases caused by pathogens (Plant and LaPatra, 2011). Available data indicates that 2 h after Vibrio anguillarum bacterin exposure, the expression of both pro-and anti-inflammatory genes increase in gilthead seabream (Sparus aurata) head kidney primary cell culture (Khansari et al., 2017). Moreover, vaccination by immersion leads to alteration of some immune genes including complement c3, tumor necrosis factor alpha (tnfα), lysozyme (lys) or transforming-growth factor beta (tgfβ) in seabream mucosal tissues such as skin and gut (Khansari et al., 2018). ...
... Thus, endocrine mobilization can also be induced by immune intermediaries since the network between the immune systems and neuroendocrine is bidirectional. At the cellular level, this network between the neuroendocrine and immune systems relies on the cells of both systems to share receptors for hormones or cytokines synthesized in both systems, thus permitting for this type of bidirectional reaction (Verburg-van Kemenade et al., 2011;Khansari et al., 2017). Ghooshchian et al. (2017) reported that Fe 3 O 4 -MNPs stimulate oxidative stress, induce apoptosis of neural cells, and change gene expression. ...
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The prevalent exposition of metallic nanoparticles (MNPs) to the aquatic medium and their negative influence on human life is one of the major concerns global. Stress mechanization, as a non-specific and pervasive response, involves all physiological systems, particularly the closely interconnected neuroendocrine and immune systems. In this study, which was designed to obtain more data on the biological effects of ulexit, which prevents oxidative DNA damage by protecting against toxicity damage and offers new antioxidant roles. The concomitant use of ulexite (UX, as 18.75 mg/L) as a natural therapeutic agent against exposure to magnetic nanoparticles (Fe3O4-MNPs/0.013 ml/L) on Oncorhynchus mykiss was investigated for 96 h. The brain tissues were taken at the 48th and 96th hours of the trial period, the effects on neurotoxic, pro-inflammatory cytokine genes, antioxidant immune system, DNA and apoptosis mechanisms were analyzed. In the present study, it was determined that AChE activity and BDNF level in the brain tissue decreased over time in the Fe3O4-MNPs group compared to the control, and UX tried to depress this inhibition. While inhibition was determined in antioxidant system biomarkers (SOD, CAT, GPx, and GSH values), an induction was observed in lipid peroxidation indicators (MDA and MPO values) in Fe3O4-MNPs applied group. The same group data showed that TNF-α, IL-6, 8-OHdG and caspase-3 levels were increased, but Nrf-2 levels were decreased. The alterations in all biomarkers were found to be significant at the p < 0.05 level. In general, it was determined that Fe3O4-MNPs caused stress in O. mykiss and UX exhibited a positive effect on this stress management.
... Cytokine genes such as TNF-α, IL-1β, IL-10, and TGF-β play important roles in the inflammatory response (Hodgkinson et al., 2015;Khansari et al., 2017;Sun et al., 2018). βAR activation is recurrently associated with the inhibition of the pro-inflammatory program and potentiated expression of anti-inflammatory factors in immune cells (Scanzano and Cosentino, 2015). ...
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... Stressors affect the activation of hypothalamus-pituitary-interrenal axis (HPI) in different organs, which is involved in immune and stress response in different species of teleost. Studies showed that although most fishes exhibit a general stress response, the pattern and magnitude of the response may be influenced by environmental factors such as ammonium, temperature, and salinity [75,76]. Similar to the results of our control setup, cortisol levels were previously found to be increased, and immune responses were decreased in changing or stressful conditions for aquatic species [8,54,72]. ...
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The aim of this study was isolation and characterization of heterotrophic bacteria capable of ammonium and nitrite removal at 15 °C (optimal temperature for growing rainbow trout Oncorhynchus mykiss). Environmental isolates were grown in liquid media containing ammonium or nitrite, and best strains in terms of growth and ammonium or nitrite removal were identified via 16S rRNA sequencing. Dyadobacter sp. (no. 68) and Janthinobacterium sp. (no. 100) were selected for optimal adaptation to growth at 15 °C and best ammonium and nitrite removal (P < 0.05), respectively. A heterotrophic ammonium and nitrite removal (HAN) microbial complex, containing selected strains, was prepared and applied in a trout culture system. After 10 days, the effect of microbial HAN complex was investigated in terms of ammonium and nitrite removal, as well as stress and immune indices present in the plasma of cultivated trout. Compared to a standard cultivation setup, addition of the HAN complex had a clear beneficial effect on keeping the un-ionized ammonia and nitrite level below prescribed standards (P < 0.05). This resulted in reduction of stress and immune reactions of cultivated fish (P < 0.05), leading to an augmentation of final weight and survival. Application of the selected microbial complex resulted in a significant improvement of the aquaculture ecosystem.
... Fish vaccinations are challenging situations that require the welltuned communication between the immune and endocrine systems to maintain a balance between the stress response and the immunological reaction to the vaccine [24][25][26][27]. The head kidney plays an important role in the immune-endocrine interaction as it combines key features of both systems: haematopoiesis, antibody production, and cortisol and catecholamine production. ...
Article
This study assessed the impact of routine vaccination of Atlantic salmon pre-smolts on gene expression and the possible link to saprolegniosis on Scottish fish farms. Fish were in 4 different groups 1) ‘control’ – fish without handling or vaccination 2) ‘vaccinated’ - fish undergoing full vaccination procedure 3) ‘non vaccinated’ - fish undergoing full vaccination procedure but not vaccinated and 4) ‘vaccinated-MH’ – fish undergoing vaccination, but procedure involved minimal handling. A strong increase in cortisol and glucose levels was observed after 1 h in all groups relative to the control group. Only in the non-vaccinated group did the level decrease to near control levels by 4 h. Expression levels of six stress marker genes in general for all groups showed down regulation over a 9-day sampling period. In contrast, expression levels for immune response genes in the head kidney showed significant up-regulation for all eight genes tested for both vaccinated groups whereas the non-vaccinated group showed up-regulation for only MHC-II and IL-6b in comparison to the control. Both the vaccination procedure and the administration of the vaccine itself were factors mediating changes in gene expression consistent with fish being susceptible to natural occurring saprolegniosis following vaccination.
... environments involving multiple stressors provoke a status of immunosuppression [6][7][8] related to severe consequences for aquaculture industry [9][10][11][12] . In essence, steroid hormones and neuroamines transfer the stress signals and impair a variety of immune functions [13][14][15] , including the reduced viability, proliferation and migration of immune-cell populations 16 , limited phagocytic activity and decreased production of pro-inflammatory mediators and antibodies [17][18][19] . These exogenously induced disturbances of the fishes' homeostasis are reflected in various physiological alterations such as changed concentrations of plasma proteins or the altered expression of specific genes. ...
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The objective of the present study is to identify and evaluate informative indicators for the welfare of rainbow trout exposed to (A) a water temperature of 27 °C and (B) a stocking density of 100 kg/m3 combined with a temperature of 27 °C. The spleen-somatic and condition index, haematocrit and the concentrations of haemoglobin, plasma cortisol and glucose revealed non-significant differences between the two stress groups and the reference group 8 days after the onset of the experiments. The transcript abundance of almost 1,500 genes was modulated at least twofold in in the spleen of rainbow trout exposed to a critical temperature alone or a critical temperature combined with crowding as compared to the reference fish. The number of differentially expressed genes was four times higher in trout that were simultaneously challenged with high temperature and crowding, compared to trout challenged with high temperature alone. Based on these sets of differentially expressed genes, we identified unique and common tissue- and stress type-specific pathways. Furthermore, our subsequent immunologic analyses revealed reduced bactericidal and inflammatory activity and a significantly altered blood-cell composition in challenged versus non-challenged rainbow trout. Altogether, our data demonstrate that heat and overstocking exert synergistic effects on the rainbow trout's physiology, especially on the immune system.
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Aquaculture conditions expose fish to internal and environmental stressors that increase their susceptibility to morbidity and mortality. The brain accumulates stress signals and processes them according to the intensity, frequency duration and type of stress, recruiting several brain functions to activate the autonomic or limbic system. Triggering the autonomic system causes the rapid release of catecholamines, such as adrenaline and noradrenaline, into circulation from chromaffin cells in the head kidney. Catecholamines trigger blood cells to release proinflammatory and regulatory cytokines to cope with acute stress. Activation of the limbic axis stimulates the dorsolateral and dorsomedial pallium to process emotions, memory, behavior and the activation of preoptic nucleus-pituitary gland-interrenal cells in the head kidney, releasing glucocorticoids, such as cortisol to the bloodstream. Glucocorticoids cause downregulation of various immune system functions depending on the duration, intensity, and type of chronic stress. As stress persists, most immune functions, with the exception of cytotoxic functions, overcome these effects and return to homeostasis. The deterioration of cytotoxic functions during chronic stress appears to be responsible for increased morbidity and mortality.
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The stress response in fish is characterized by the activation of the HPI axis resulting in the release of cortisol. Previous studies in rainbow trout (Oncorhynchus mykiss) and gilthead sea bream (Sparus aurata) have shown that an acute stressor modulates the expression of immune- and stress-related genes in mucosal-associated lymphoid tissues (MALTs), particularly in the skin (SALT), gills (GIALT), and gut (GALT). However, there are no antecedents on whether the modulation on the mucosal transcriptomic profile is coordinated through the local presence of cortisol in the mucosal tissue surface. Thus, the aim of this study was to evaluate the effect of cortisol upon the modulation of a set of immune- (il-1β, il-6, tnf-α, and cox-2) and stress-related (hsp70, gr1) genes. For this purpose, tissue explants cultures were incubated with cortisol (100 ng/100 mg tissue) for 2-, 4-, and 24 h and the gene expression profile was evaluated at each time-point by real-time PCR. No differences were found in the gene expression between cortisol-incubated tissue explants and mock-incubated tissues in any of the time-points tested for both species. These results suggest that the quick modulation of the gene expression during the first 24 h after the exposure to stressor challenge reported in previous studies, is probably coordinated and mediated through a systemic-dependent mechanism but not through a peripheral/local response on mucosal tissue surfaces.
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Over 60 years ago, Selye1 recognized the paradox that the physiologic systems activated by stress can not only protect and restore but also damage the body. What links these seemingly contradictory roles? How does stress influence the pathogenesis of disease, and what accounts for the variation in vulnerability to stress-related diseases among people with similar life experiences? How can stress-induced damage be quantified? These and many other questions still challenge investigators. This article reviews the long-term effect of the physiologic response to stress, which I refer to as allostatic load.2 Allostasis — the ability to achieve stability through change3 — . . .
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The secretion of glucocorticoids (GCs) is a classic endocrine response to stress. Despite that, it remains controversial as to what purpose GCs serve at such times. One view, stretching back to the time of Hans Selye, posits that GCs help mediate the ongoing or pending stress response, either via basal levels of GCs permitting other facets of the stress response to emerge efficaciously, and/or by stress levels of GCs actively stimulating the stress response. In contrast, a revisionist viewpoint posits that GCs suppress the stress response, preventing it from being pathologically overactivated. In this review, we consider recent findings regarding GC action and, based on them, generate criteria for determining whether a particular GC action permits, stimulates, or suppresses an ongoing stress-response or, as an additional category, is preparative for a subsequent stressor. We apply these GC actions to the realms of cardiovascular function, fluid volume and hemorrhage, immunity and inflammation, metabolism, neurobiology, and reproductive physiology. We find that GC actions fall into markedly different categories, depending on the physiological endpoint in question, with evidence for mediating effects in some cases, and suppressive or preparative in others. We then attempt to assimilate these heterogeneous GC actions into a physiological whole.
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To review results on exercise induced changes in the immune system following strenuous and moderate exercise. A literature search over the past 15 years was conducted using Medline and selected papers. After intense long term exercise, the immune system is characterised by concomitant impairment of the cellular immune system and increased inflammation. Thus low concentrations of lymphocytes, suppressed natural immunity, suppressed lymphocyte proliferation, and suppressed levels of secretory IgA in saliva are found simultaneously with high levels of circulating proinflammatory and antiinflammatory cytokines. The underlying mechanisms are multifactorial and include neuroendocrinological and metabolic factors. The clinical consequences of the exercise induced immune changes have not formally been identified, but the exercise effect on lymphocyte dynamics and immune function may be linked to the exercise effects on resistance to infections and malignancy and the cytokine response may be linked to muscle damage or muscle cell growth. Moderate exercise across the life span seems to increase resistance to upper respiratory tract infections, whereas repeated strenuous exercise suppresses immune function. It is premature to offer advice on nutrition to athletes in order to alter the exercise induced immunosuppression found after exercise.
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A tight interaction between endocrine and immune systems takes place mainly due to the key role of head kidney in both hormone and cytokine secretion, particularly under stress situations in which the physiological response promotes the synthesis and release of stress hormones which may lead into immunomodulation as side effect. Although such interaction has been previously investigated, this study evaluated for the first time the effect of stress-associated hormones together with their receptor antagonists on the expression of cytokine genes in head kidney primary cell culture (HKPCC) of the freshwater rainbow trout (Oncorhynchus mykiss) and the seawater gilthead sea bream (Sparus aurata). The results showed a striking difference when comparing the response obtained in trout and seabream. Cortisol and adrenocorticotropic hormone (ACTH) decreased the expression of immune-related genes in sea bream but not in rainbow trout and this cortisol effect was reverted by the antagonist mifepristone but not spironolactone. On the other hand, while adrenaline reduced the expression of pro-inflammatory cytokines (IL-1β, IL-6) in rainbow trout, the opposite effect was observed in sea bream showing an increased expression (IL-1β, IL-6). Interestingly, this effect was reverted by antagonist propranolol but not phentolamine. Overall, our results confirm the regional interaction between endocrine and cytokine messengers and a clear difference in the sensitivity to the hormonal stimuli between the two species.
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1. Introduction2. Effects of Stressors on the Immune Response 2.1. Suppressive Versus Enhancing Effects2.2. Perception of Stress After Immune Stimulation: Systemic Versus Local Responses2.3. Stress and the Cellular and Humoral Immune Response3. Organization of the Immune Response Following Stress: The Neuroimmunoendocrine Connection and the Role of the Head Kidney4. Effects of Hormones on the Immune System 4.1. Hypothalamic Hormones4.2. Pituitary Hormones4.3. Interrenal Hormones4.4. Receptor-Mediating Action of Cortisol in Fish Immunity During Stress Response4.5. Somatotropic Axis and Fish Immune System5. Environmental Stressors and Fish Immunity 5.1. Environmental Salinity5.2. Temperature and Seasonality6. Future DirectionsThe endocrine-immune relationship of fish, particularly related to the stress response, is mediated by the close interaction of hormones and cytokines. In essence, stress can depress certain elements of the immune system and render fish vulnerable to infection and disease. This chapter summarizes the effects of stressors on disease resistance and the immune system and updates the knowledge on endocrine regulation of the immune system in fish, the effects at systemic and local levels, and the organization of the immune responses under stressed conditions, with special emphasis on the roles of hormones, their receptors, and system interactions. Basically, low levels of severity of stress (eustress) may lead to enhanced immune competence while greater severities tend to be immunosuppressive. The immune response to stressors are mediated by the endocrine system at both central and peripheral levels.
Article
In the last years, the aquaculture crops have experienced an explosive and intensive growth, because of the high demand for protein. This growth has increased fish susceptibility to diseases and subsequent death. The constant biotic and abiotic changes experienced by fish species in culture are challenges that induce physiological, endocrine and immunological responses. These changes mitigate stress effects at the cellular level to maintain homeostasis. The effects of stress on the immune system have been studied for many years. While acute stress can have beneficial effects, chronic stress inhibits the immune response in mammals and teleost fish. In response to stress, a signaling cascade is triggered by the activation of neural circuits in the central nervous system because the hypothalamus is the central modulator of stress. This leads to the production of catecholamines, corticosteroid-releasing hormone, adrenocorticotropic hormone and glucocorticoids, which are the essential neuroendocrine mediators for this activation. Because stress situations are energetically demanding, the neuroendocrine signals are involved in metabolic support and will suppress the "less important" immune function. Understanding the cellular mechanisms of the neuroendocrine regulation of immunity in fish will allow the development of new pharmaceutical strategies and therapeutics for the prevention and treatment of diseases triggered by stress at all stages of fish cultures for commercial production.
Article
Relatively little is known about factors that modulatein vivolower vertebrate phagocyte responses, particularly in fish. In this study we report that head-kidney leucocytes from the marine fish gilthead seabream (Sparus aurataL.) release soluble macrophage-activating factor/s (MAF) after stimulation with concanavalin A (Con A) and phorbol myristate acetate (PMA). Migration, phagocytosis, reactive oxygen intermediates (ROI) production and bactericidal activity were enhanced after incubation of target phagocytes with the MAF-containing supernatants. In order to ascertain the effect of MAF and bacterial lipopolysaccharide (LPS) on ROI and reactive nitrogen intermediates (RNI) production in gilthead seabream macrophages, macrophages were incubated with MAF in the presence of LPS. The results revealed that co-stimulation of macrophages with MAF and LPS inhibited O2−production but synergistically induced release of nitric oxide. These data suggest that the main activities of fish phagocytes are regulated by cytokines, which can interact positively or negatively with pathogen-derived molecules to determine the final activation state of the cells.
Article
Live attenuated vaccines are a promising application to control bacterial fish diseases. A live attenuated Vibrio anguillarum vaccine candidate was established in our laboratory to protect fish against vibriosis. To elucidate the mechanism of immunoprotection, it is necessary to compare the different immune responses to infection between vaccinated and non-vaccinated fish. In this study, the expression levels of pathogen-specific antibodies and immune-related genes upon challenge at 28 days post-vaccination were compared between vaccinated and non-vaccinated zebrafish. In the results, the specific antibody levels against virulent V. anguillarum in the vaccinated group did not rise significantly following infection, which suggested that high-affinity antibodies were induced by the vaccine. In the non-vaccinated group, the specific IgM response was triggered at 3 days post-infection and showed a delayed antibody response. Meanwhile, the transcription levels of the genes encoding the pro-inflammatory cytokine IL-1β and the chemokine IL-8 were more highly up-regulated in non-vaccinated fish than in vaccinated fish. This suggests that the overwhelming inflammatory response trigged by infection in non-vaccinated zebrafish was controlled in vaccinated zebrafish. Interestingly, the expression levels of adaptive immune-related genes were increased in vaccinated fish after challenge, compared to the non-vaccinated fish. These results suggest that inoculation with the live attenuated vaccine triggered protection by curbing inflammation and strengthening the adaptive immune response.
Article
Cells from goldfish and amphibian lymphoid organs, mainly leukocytes, express high affinity β-adrenergic receptors specific for β-adrenergic ligands (agonists: adrenaline, noradrenaline, terbutaline, and fenoterol; antagonists: CGP-12177, dihydroalprenolol, propranolol, atenolol, and butoxamine). The rank order of ligand potency does not allow their being classified into any known mammalian subtype. Among features that distinguish them from mammalian β1 and β2-adrenoceptors is much lower affinity for (-)-CGP-12177, obtained in both saturation and kinetic experiments (about 25 nM for goldfish head kidney cells). The density of receptors on goldfish and anuran cells is organ-dependent and comparable to that estimated on mammalian leukocytes. The extraordinarily high receptor density on salamander splenic cells (about 183,000) correlates with the large size of urodele cells. The competition experiments on goldfish cells with propranolol and CGP-12177 suggest the existence of yet another binding site, which may be either another β-AR subtype, or a serotonergic receptor.
Article
It has long been held that cortisol, a glucocorticoid in many vertebrates, performs glucocorticoid and mineralocorticoid actions in the teleost fish since it lacks aldosterone. However, in addition to the counterparts of tetrapod mineralocorticoid receptors (MRs), 11-deoxycorticosterone (DOC) has been recently identified as a specific endogenous ligand for the MRs in teleosts. Here, we point out the minor role of mineralocorticoid signaling (i.e., DOC-MR) in the osmoregulation compared with those of glucocorticoid signaling (i.e., cortisol-glucocorticoid receptor [GR]), and review the current findings on the physiological roles of the DOC-MR in teleosts. Cortisol promotes both freshwater and seawater adaptation via the GRs in the osmoregulatory organs such as gills and gastrointestinal tracts, but the expressions of MR mRNA are abundant in the brains especially in the key components of the stress axis and cerebellums. Together with the behavioral effects of intracerebroventricular injection with DOC, the MR is suggested to play an important role in the brain dependent behaviors. Since the abundant expression of central MRs has been reported also in higher vertebrates and the MR is thought to be ancestral to the GR, the role of MR in fish might reflect the principal and original function of corticosteroid signaling. Functional evolution of corticosteroid systems is summarized and areas in need of research like our on-going experiments with MR-knockout medaka are outlined.
Article
Inflammatory responses have to be carefully controlled, as high concentrations and/or prolonged action of inflammation-related molecules (e.g. reactive oxygen species, nitric oxide and pro-inflammatory cytokines) can be detrimental to host tissue and organs. One of the potential regulators of the inflammatory process are stress mediators including adrenaline. In vivo effects of adrenaline were studied during zymosan-induced (Z) peritoneal inflammation in the common carp Cyprinus carpio L. Adrenaline injected together with zymosan (ZA) did not change the number of inflammatory leukocytes in the peritoneal cavity, however at 24 h post-injection it significantly reduced the percentage of monocytes/macrophages. Moreover, compared to cells retrieved from fish treated with PBS or zymosan only, adrenaline increased the percentage of apoptotic leukocytes in the focus of inflammation. Furthermore, adrenaline significantly reduced the expression of chemokine CXCa (a functional homolog of mammalian IL-8) and its receptors (CXCR1 and CXCR2), indicating changes in leukocyte recruitment after stress. We conclude that adrenaline may contribute to a coordinated reaction by influencing the inflammatory response via direct regulation of leukocyte migration and/or apoptosis.
Article
Although liver is a key target for corticosteroid action, its role in immune function is largely unknown. We tested the hypothesis that stress levels of cortisol down regulate immune-relevant genes in rainbow trout (Oncorhynchus mykiss) liver. Hepatocytes were treated with lipopolysaccharide (LPS) for 24h either in the presence or absence of cortisol. LPS stimulated heat shock protein 70 expression, enhanced glycolytic capacity, and reduced glucose output. LPS stimulated mRNA abundance of cytokines and serum amyloid protein A (SAA), while suppressors of cytokine signaling (SOCS)-3 was reduced. Cortisol increased mRNA abundances of IL-1β, SOCS-1 and SOCS-2, while inhibiting either basal or LPS-stimulated IL-8, TNF α2 and SAA. These cortisol-mediated effects were rescued by Mifepristone, a glucocorticoid receptor antagonist. Altogether, cortisol modulates the molecular immune response in trout hepatocytes. The upregulation of SOCS-1 and SOCS-2 by cortisol may be playing a key role in suppressing cytokine signaling and the associated inflammatory response.
Article
The sea louse, Lepeophtheirus salmonis, is an ectoparasitic copepod of Atlantic salmon, Salmo salar L., capable of causing severe damage. This study was conducted to examine the physiological response of salmon to the stress of sea lice infestation. Smoltified salmon were acclimatized in 30‰ saltwater and exposed to high levels of lice infestation. The number of copepods per fish ranged from 15 to 285, with a mean of 106. The infested salmon were sampled six times over the 29-d experimental duration and examined for alterations in the primary and secondary stress indicators, including plasma concentrations of cortisol, glucose, electrolytes, thyroid hormones T3 and T4, as well as the haematocrit level. The results were examined for correlations between the stress indicators, the number of copepods per fish and the life stage of the copepods. The presence of L. salmonis elevated stress indicators in relation to the specific sea lice stage. By day 21, both cortisol (mean 63.1 nmol L−1 controls: 179.8 nmol L−1 for parasitized) and glucose (mean 3.545 mmol L−1 controls: 4.567 mmol L−1 for parasitized) levels were significantly increased due to the presence of the lice. This was believed to be a direct result of the sea lice development into the larger life stages, thus increasing the level of host damage.
Article
Vibrio angillarum is an important pathogen for large yellow croaker, being responsible for significant economic losses in Chinese aquaculture. Gene expression was studied in large yellow croaker stimulated with an attenuated live V. anguillarum MVAV6203. Subtractive cDNA libraries were constructed by suppression subtractive hybridization (SSH) from 3 immune-relevant tissues during the process. Approximately 216 clones were sequenced from the library, giving a total of 179 different expressed sequence tags (ESTs), which were further identified and annotated by comparing with the existing genes in GenBank. These identified ESTs represented at least 39 different genes which are involved in immune system, cell communication, protein regulation and other processes, most of which are not previously reported in large yellow croaker. Expression of 4 selected genes encoding complement component inhibitor 1, hepcidin, α-NAC and coagulation factor II in liver, spleen and head kidney of the fish was further studied by real-time quantitative PCR to verify their differential expression as a result of induction. A clear increase in expression in liver and head kidney of the fish between challenged and control samples was observed for genes encoding complement component inhibitor 1 and hepcidin, which seems to play an important role in immune system of fishes.
Article
Atlantic salmon were vaccinated by intraperitoneal injection of particulate lipopolysaccharide (LPS) antigens of the two fish pathogens Vibrio salmonicida and Vibrio anguillarum. Particulate LPS from V. salmonicida and V. anguillarum serotype 01 failed to demonstrate a protection against disease after intraperitoneal challenge with live bacteria. However, fish vaccinated with particulate LPS preparations from V. anguillarum serotype 02 acquired a high protection and the LPS-protein complex surface layer antigen VS-P1 from V. salmonicida was seen to give a protection which was superior to purified LPS alone. Vaccination with LPS particles modified by precoating with bovine serum albumin or oleic acid resulted in a slightly better protection compared to the unmodified LPS particle.
Article
Cortisol, the primary circulating corticosteroid in teleosts, is elevated during stress following activation of the hypothalamus-pituitary-interrenal (HPI) axis. Cortisol exerts genomic effects on target tissues in part by activating glucocorticoid receptors (GR). Despite a well-established negative feedback loop involved in plasma cortisol regulation, the role of GR in the functioning of the HPI axis during stress in fish is still unclear. We used mifepristone (a GR antagonist) to suppress GR signaling in rainbow trout (Oncorhynchus mykiss) and assessed the resultant changes to HPI axis activity. We show for the first time that mifepristone caused a functional knockdown of GR by depleting protein expression 40-75%. The lower GR protein expression corresponded with a compensatory up-regulation of GR mRNA levels across tissues. Mifepristone treatment completely abolished the stressor-induced elevation in plasma cortisol and glucose levels seen in the control fish. A reduction in corticotropin-releasing factor (CRF) mRNA abundance in the hypothalamic preoptic area was also observed, suggesting that GR signaling is involved in maintaining basal CRF levels. We further characterized the effect of mifepristone treatment on the steroidogenic capacity of interrenal tissue in vitro. A marked reduction in cortisol production following adrenocorticotropic hormone stimulation of head kidney pieces was observed from mifepristone treated fish. This coincided with the suppression of steroidogenic acute regulatory protein, but not P450 side chain cleavage mRNA abundances. Overall, our results underscore a critical role for central and peripheral GR signaling in the regulation of plasma cortisol levels during stress in fish.
Article
The majority (91%) of 260 isolates initially identified as Vibrio anguillarum, that were obtained from a wide range of hosts, habitats and geographical locations, were recovered in a single cluster based on the ribotype and were pathogenic to Atlantic salmon. A significant proportion of isolates (78% of the total) were allocated to 15 serogroups (O1–O10 and five previously undescribed groups referred to as VaNT1, VaNT2, VaNT4, NaNT5 and VaNT7). A minority of isolates (6%) reacted with more than one antiserum or were self-agglutinating, and the remainder did not react with any of the antisera tested. Good correlation was noted between serogroups and lipopolysaccharide profiles, particularly with respect to isolates belonging to serogroups O1, 02 and 04ߝ010. Plasmids were recognized in some serogroups. especially O1, which contained the 67 kb plasmid associated with virulence. However, the 19 profiles based on outer membrane protein patterns did not correspond to the results obtained with the other typing methods. Generally, the isolates were heterogeneous in their biochemical characteristics; 117 profiles were obtained with the API 20E system, and 9 and 32 clusters recognised from the results of BIOLOG fingerprinting and Biotype-100 biotyping methods, respectively. Three dominant dusters were defined from fatty acid methyl esters profiles.
Article
Stress is an event that most animals experience and that induces a number of responses involving all three regulatory systems, neural, endocrine and immune. When the stressor is acute and short-term, the response pattern is stimulatory and the fish immune response shows an activating phase that specially enhances innate responses. If the stressor is chronic the immune response shows suppressive effects and therefore the chances of an infection may be enhanced. In addition, coping with the stressor imposes an allostatic cost that may interfere with the needs of the immune response. In this paper the mechanisms behind these immunoregulatory changes are reviewed and the role of the main neuroendocrine mechanisms directly affecting the building of the immune response and their consequences are considered.
Article
Delayed type hypersensitivity (DTH) reactions are antigen-specific, cell-mediated immune responses which, depending on the antigen involved, mediate beneficial (resistance to viruses, bacteria, fungi, and certain tumors) or harmful (allergic dermatitis, autoimmunity) aspects of immune function. We have shown that acute stress administered immediately before antigenic challenge results in a significant enhancement of a skin DTH response in rats. A stress-induced trafficking or redeployment of leukocytes to the skin may be one of the factors mediating this immunoenhancement. Here we investigate the effects of varying the duration, intensity, and chronicity of stress on the DTH response and on changes in blood leukocyte distribution and glucocorticoid levels. Acute stress administered for 2 h prior to antigenic challenge, significantly enhanced the DTH response. Increasing the duration of stress from 2 h to 5 h produced the same magnitude enhancement in cutaneous DTH. Moreover, increasing the intensity of acute stress produced a significantly larger enhancement of the DTH response which was accompanied by increasing magnitudes of leukocyte redeployment. In contrast, chronic stress suppressed the DTH response when it was administered for 3 weeks before sensitization and either discontinued upon sensitization, or continued an additional week until challenge, or extended for one week after challenge. The stress-induced redeployment of peripheral blood lymphocytes was attenuated with increasing exposure to chronic stress and correlated with attenuated glucocorticoid responsivity. These results suggest that stress-induced alterations in lymphocyte redeployment may play an important role in mediating the bi-directional effects of acute versus chronic stress on cell-mediated immunity in vivo.
Article
Catecholamines exert their physiological actions through α and β adrenergic receptors (ARs). As ARs are not exclusively expressed on neuroendocrine cells, but also on leukocytes, they may facilitate neuroendocrine modulation of immune responses. We sequenced the β(2a)-AR in common carp, and studied its expression profile and involvement in the regulation of teleost innate immune responses. β(2a)-AR messenger RNA was found to be constitutively expressed in brain areas, especially in the preoptic nucleus (NPO, homologous to the mammalian hypothalamus), and in immune organs. During the active phase of an in vivo inflammatory response, induced by i.p. zymosan treatment, β(2a)-AR gene expression was up-regulated in the peritoneal leukocytes. Additionally, adrenaline in vitro reduced the synthesis of oxygen radical species and nitric oxide, while it enhanced arginase activity in fish phagocytes. Furthermore, in vitro adrenaline administration inhibited expression of pro-inflammatory cytokines, chemokines and their receptors. It is therefore hypothesized that adrenaline will down-regulate phagocyte skewing toward classical/innate polarization.
Article
Glucocorticoid actions on the immune system are diverse and cell type dependent, and little is known about cell type-specific interactions and cross-talk between hormones and cytokines. In this study we have analyzed the gene expression patterns of the rainbow trout macrophage cell line RTS-11 by quantitative PCR, after exposure to combinations of cortisol plus a pro-inflammatory cytokine (e.g. recombinant trout IL-1β, IFN-γ), type I IFN or a PAMP (LPS or poly I:C). Several key genes of the inflammatory process were targetted to assess whether any modulation of their expression occurred due to the addition of cortisol to this cell line. Incubation of macrophages for 3 or 6 h with a physiological concentration of cortisol caused a decrease in expression of IL-6 and IL-8, but no significant changes were observed for the other genes examined. Co-stimulation of cortisol with the inflammatory agents resulted in a general suppression of genes related to the inflammatory response. Cortisol inhibited the up-regulation of IL-8 by all the stimulants after 3 h of co-incubation. Suppression of the up-regulation of IL-6 by rIL-1β, rIFN-γ and poly I:C, of γIP by rIFN-γ or poly I:C, and of Cox-2 by rIL-1β was seen after 6 h. In contrast, cortisol in combination with the pro-inflammatory agents has a synergistic effect on IL-10 expression, an anti-inflammatory molecule, suggesting that the activation of certain macrophage functions that lead to the resolution of inflammation occurs in fish macrophages in response to cortisol treatment.
Article
The discovery of Toll-like receptors (TLRs) as components that recognize conserved structures in pathogens has greatly advanced understanding of how the body senses pathogen invasion, triggers innate immune responses and primes antigen-specific adaptive immunity. Although TLRs are critical for host defense, it has become apparent that loss of negative regulation of TLR signaling, as well as recognition of self molecules by TLRs, are strongly associated with the pathogenesis of inflammatory and autoimmune diseases. Furthermore, it is now clear that the interaction between TLRs and recently identified cytosolic innate immune sensors is crucial for mounting effective immune responses. Here we describe the recent advances that have been made by research into the role of TLR biology in host defense and disease.
Article
Neuro-endocrine and immune systems closely interact in fish, and their regulation is crucial for the maintenance of good health of cultured fish. We have used the seabream head kidney to study whether stress-related hormones can modulate the immune response. For this purpose, the effects of adrenaline, adrenocorticotropic hormone (ACTH) and cortisol on the expression of pro-inflammatory cytokines (TNF-alpha, IL-1beta, IL-6) and the anti-inflammatory cytokine TGF-beta1 were determined by means of quantitative real-time PCR on isolated head kidney cells. ACTH (150 ng mL(-1)) caused an acute increase of TNF-alpha and IL-6 mRNA levels as well as an inhibition of IL-1beta expression. The expression of the anti-inflammatory cytokine TGF-beta1 was also increased, although in a lower extent. Adrenaline (1 muM) early effects were only clear inhibiting IL-1beta expression but not TNF-alpha, IL-6 or TGF-beta1 mRNA levels, while a longer exposure to the hormone inhibited all cytokines. Moreover, cortisol (50 and 100 ng mL(-1)) reduced the expression of all cytokines in a dose-dependent manner. Bacterial lipopolysaccharide (LPS) stimulated IL-1beta expression and inhibited that of the anti-inflammatory TGF-beta1, although it was ineffective on TNF-alpha and IL-6. In addition, adrenaline and cortisol decreased the LPS-stimulated IL-1beta expression, further demonstrating their previously reported anti-inflammatory effects. The combination of ACTH and LPS, on the other hand, did not affect LPS-stimulated IL-1beta expression but was effective increasing TNF-alpha expression. Taking all these results in consideration, we conclude that the expression of pro- and anti-inflammatory cytokines in the seabream head kidney is highly influenced by stress-related hormones, thus indicating an important role for the endocrine system in the modulation of the immune response in teleost fish.
Article
Stress is known to suppress immune function and increase susceptibility to infections and cancer. Paradoxically, stress is also known to exacerbate asthma, and allergic, autoimmune and inflammatory diseases, although such diseases should be ameliorated by immunosuppression. Moreover, the short-term fight-or-flight stress response is one of nature's fundamental defense mechanisms that enables the cardiovascular and musculoskeletal systems to promote survival, and it is unlikely that this response would suppress immune function at a time when it is most required for survival (e.g. in response to wounding and infection by a predator or aggressor). These observations suggest that stress may suppress immune function under some conditions while enhancing it under others. The effects of stress are likely to be beneficial or harmful depending on the type (immunoprotective, immunoregulatory/inhibitory, or immunopathological) of immune response that is affected. Studies have shown that several critical factors influence the direction (enhancing vs. suppressive) of the effects of stress or stress hormones on immune function: (1) Duration (acute vs. chronic) of stress: Acute or short-term stress experienced at the time of immune activation can enhance innate and adaptive immune responses. Chronic or long-term stress can suppress immunity by decreasing immune cell numbers and function and/or increasing active immunosuppressive mechanisms (e.g. regulatory T cells). Chronic stress can also dysregulate immune function by promoting proinflammatory and type-2 cytokine-driven responses. (2) Effects of stress on leukocyte distribution: Compartments that are enriched with immune cells during acute stress show immunoenhancement, while those that are depleted of leukocytes, show immunosuppression. (3) The differential effects of physiologic versus pharmacologic concentrations of glucocorticoids, and the differential effects of endogenous versus synthetic glucocorticoids: Endogenous hormones in physiological concentrations can have immunoenhancing effects. Endogenous hormones at pharmacologic concentrations, and synthetic hormones, are immunosuppressive. (4) The timing of stressor or stress hormone exposure relative to the time of activation and time course of the immune response: Immunoenhancement is observed when acute stress is experienced at early stages of immune activation, while immunosuppression may be observed at late stages of the immune response. We propose that it is important to study and, if possible, to clinically harness the immunoenhancing effects of the acute stress response, that evolution has finely sculpted as a survival mechanism, just as we study its maladaptive ramifications (chronic stress) that evolution has yet to resolve. In view of the ubiquitous nature of stress and its significant effects on immunoprotection as well as immunopathology, it is important to further elucidate the mechanisms mediating stress-immune interactions and to meaningfully translate findings from bench to bedside.
Article
Stress has profound effects on vertebrate immunity, but most studies have considered stress-immune interactions in terms of wild animals enduring demanding, but predictable activities (e.g., immune alterations during breeding). A growing biomedical literature, however, indicates that stress may not be obligatorily immunosuppressive; in response to transient, unpredictable stressors, immune activity can be enhanced, especially in body areas requiring immune protection. Also, immune sensitivity to stressors is not fixed throughout life; oftentimes, glucocorticoid (GC) insensitivity can be induced. Further GC sensitivity can be programmed early in life; greater exposure to stressors prior to maturity heightens GC effects on immunity in adulthood. In the present paper, I review the cellular and molecular mechanisms that link stress responses to immune adjustments over short time scales in domesticated species then I attempt to place stress-immune interactions in a naturalistic, organismal context. When, how and why stressors affect immunity in wild animals remains practically unstudied.
Article
The stress hormone cortisol is deeply involved in immune regulation in all vertebrates. Common carp (Cyprinus carpio L.) express four corticoid receptors that may modulate immune responses: three glucocorticoid receptors (GR); GR1, with two splice variants (GR1a and GR1b), GR2 and a single mineralocorticoid receptor (MR). All receptors are expressed as of 4 days post-fertilization and may thus play a critical role in development and functioning of the adult immune system. Immune tissues and cells predominantly express mRNA for GRs compared to mRNA for the MR. Three-dimensional protein structure modeling predicts, and transfection assays confirm that alternative splicing of GR1 does not influence the capacity to induce transcription of effector genes. When tested for cortisol activation, GR2 is the most sensitive corticoid receptor in carp, followed by the MR and GR1a and GR1b. Lipopolysacharide (LPS) treatment of head kidney phagocytes quickly induces GR1 expression and inhibits GR2 expression. Cortisol treatment in vivo enhances GR1a and MR mRNA expression, but only mildly, and cortisol treatment in vitro does not affect receptor expression of phagocytes. Cortisol has no direct effect on the LPS-induced receptor profile. Therefore, an immune rather than a stress stimulus regulates GR expression. Cortisol administered at stress levels to phagocytes in vitro significantly inhibits LPS-induced expression of the pro-inflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-12 (IL-12) (subunit p35) and of inducible nitric oxide synthase (iNOS) expression. A physiologically differential function for GR1 and GR2 in the immune response of fish to infection is indicated.
Article
The murine B cell line CH12.LX.C4.5F5 (CH12 (5F5) expresses adrenocorticotropin (ACTH) receptors, which can modulate IgM secretion by these cells. Interestingly, the response to ACTH was concentration dependent, inducing IgM secretion at subnanomolar amounts and suppressing secretion at micromolar amounts. With the use of an enzyme-linking immunospot assay it was possible to demonstrate that the ACTH-induced increase in IgM secretion by CH12 (5F5) cells was caused at least in part by an increase in the number of cells secreting IgM. CH12 (5F5) cells activated with suboptimal concentrations of LPS demonstrated a similar biphasic response. ACTH at concentrations of 10(-13) to 10(-9) M augmented IgM secretion in LPS-activated cells as much as sixfold, whereas 10(-6) M ACTH slightly decreased LPS-induced IgM secretion. At the mRNA level, subnanomolar concentrations of ACTH increased microH chain mRNA expression up to twofold in unstimulated or LPS-stimulated CH12 (5F5) cells. Taken together, these studies show that physiologically relevant concentrations of ACTH can interact directly with receptors on these B lymphocytes to enhance IgM secretion and microH chain mRNA expression. Although ACTH does increase intracellular cAMP levels in CH12 (5F5) B cells, it is unlikely that the induction of this second messenger pathway is by itself responsible for the ACTH induced B cell differentiation. The concentration of ACTH necessary to stimulate significant intracellular cAMP increases was 10- to 100-fold higher than that required to increase IgM secretion. Furthermore, CH12 (5F5) cells treated with varying concentrations of 8-bromo cAMP or cholera toxin were inhibited in their ability to secrete IgM. These results strongly suggest that the enhancing effects of ACTH on CH12 (5F5) IgM secretion are via mechanisms independent of those mediated by cAMP.
Article
Treatment of lymphocytes with inducers of interferon alpha (IFN-alpha) results in the production of corticotropin (ACTH) and endorphin-like activities. The pro-opiomelanocortin-derived hormones ACTH and alpha-, beta-, and gamma-endorphin and the structurally related hormones [Leu]- and [Met]enkephalin were therefore tested for their effects on the in vitro antibody response of mouse spleen cells. ACTH and alpha-endorphin were potent inhibitors (>/=80% suppression) of the antibody response to the T-cell-dependent antigen sheep erythrocytes at a concentration of 0.5 muM. [Met]- and [Leu]enkephalin were moderate inhibitors (approximately 60% suppression) at 0.2-2 muM, and beta- and gamma-endorphin were minimal inhibitors (approximately 20% suppression) at 5-6 muM. At higher concentrations ACTH also inhibited the antibody response to the T-cell-independent antigen dinitrophenyl-Ficoll, suggesting that T-cell function was more sensitive to blockage by these hormones than was B-cell function. ACTH and IFN had similar suppression properties; thus, the hormone-like activities associated with IFN-alpha may play a role in IFN-induced immunosuppression. alpha-Endorphin immunosuppression was blocked by naloxone, which suggested that alpha-endorphin exerted its effects through binding to opiate-like receptors on the spleen cells. The failure of beta-endorphin to suppress the immune response significantly was not due to its failure to bind to the opiate-like receptors because it blocked alpha-endorphin-induced suppression. Direct evidence for both opiate and ACTH receptors on the spleen cells was obtained in binding studies with labeled enkephalin and ACTH. Such studies revealed the presence of both high- and low-affinity receptors. The data show that neuroendocrine polypeptide hormones can regulate the immune response.
Article
Primary and secondary lymphoid organs are innervated extensively by noradrenergic sympathetic nerve fibers. Lymphocytes, macrophages, and other cells of the immune system bear functional adrenoreceptors. Norepinephrine fulfills criteria for neurotransmission with cells of the immune system as targets. In vitro, adrenergic agonists can modulate all aspects of an immune response (initiative, proliferative, and effector phases), altering such functions as cytokine production, lymphocyte proliferation, and antibody secretion. In vivo, chemical sympathectomy suppresses cell-mediated (T helper-1) responses, and may enhance antibody (T helper-2) responses. Noradrenergic innervation of spleen and lymph nodes is diminished progressively during aging, a time when cell-mediated immune function also is suppressed. In animal models of autoimmune disease, sympathetic innervation is reduced prior to onset of disease symptoms, and chemical sympathectomy can exacerbate disease severity. These findings illustrate the importance of the sympathetic nervous system in modulating immune function under normal and disease states.
Article
A bidirectional communication exists between the nervous system and the immune system. Evidence has accumulated suggesting that cytokines-immune peptides influence sympathetic neuronal survival and that cytokines can promote the secretion of catecholamines. Using an isolated perfused rat liver (IPRL) preparation, we have shown that the liver is an important source of circulating cytokines in response to lipopolysaccharide (LPS) and that corticosterone dose dependently influenced LPS-induced production of tumor necrosis factor (TNF) and interleukin-6 (IL-6). In this study, we investigated the direct effect of epinephrine (another stress hormone) on the production of TNF and IL-6 in liver. We demonstrated that epinephrine (1 microM/ml) alone did not induce TNF bioactivity but significantly increased IL-6 bioactivity from IPRL effluent. When the IPRL was infused with LPS, epinephrine significantly decreased TNF bioactivity. Epinephrine in LPS-treated livers also significantly increased IL-6 bioactivity. Both responses were totally inhibited by the beta-blocker propranolol (10 microM/ml). Anisomycin, a protein synthesis inhibitor, infused into the IPRL completely blocked the rise in TNF and IL-6 concentrations in the effluent leaving the IPRL, supporting the hypothesis that the synthesis (or release) of these cytokines was dependent on protein synthesis. We then attempted to determine whether epinephrine exerts similar effects in vitro. Using isolated Kupffer cells and hepatocytes, we found that epinephrine alone had no effect on TNF and IL-6 production in Kupffer cells and hepatocytes but significantly decreased LPS-induced TNF bioactivity and increased LPS-induced IL-6 bioactivity in Kupffer cells. Our data support the hypothesis that epinephrine can promote IL-6 secretion from IPRL.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
Histocompatible Fischer 344 (F344) and Lewis (LEW) rats provide a comparative model for investigating the interactions between the nervous, endocrine and immune systems. The outbred Sprague-Dawley (SD) is the maternal strain for the inbred F344 and LEW strains. In this study we report large differences in the diurnal and stress corticosterone (CORT) profiles of these three genetically related strains: (1) F344 rats had significantly higher diurnal and stress CORT levels than SD and LEW rats; (2) in the morning, stress CORT levels of SD and F344 rats returned towards basal 1 h after cessation of the stressor, whereas stress CORT levels of LEW rats had not returned to basal by this time; and (3) in the evening, SD and F344 rats showed the expected evening rise in basal CORT levels, whereas LEW rats failed to show this rise. In light of the large differences in CORT levels, we expected to observe strain differences in absolute levels of Type I (mineralocorticoid) and Type II (glucocorticoid) adrenal steroid receptors in neural as well as immune tissue. However, we found no significant strain differences in levels of Type I receptors in the hippocampus, hypothalamus, pituitary, thymus, spleen and peripheral blood mononuclear cells. Similarly, we saw no significant strain differences in levels of Type II receptors in most of the tissues surveyed, with the notable exception that LEW rats showed higher Type II binding in the thymus, and SD rats showed small, but significantly higher Type II binding in the hippocampus. We also studied strain differences in levels of corticosteroid-binding globulin (CBG). F344 rats expressed significantly higher CBG levels than SD and LEW rats, in plasma, spleen and thymus. Future studies will investigate whether the substantial differences between strains in levels of CORT and CBG, in the context of few strain differences in post-adrenalectomy adrenal steroid receptor levels in neural and immune tissue, translate into differences in receptor occupancy/activation under resting conditions, or following stress.
Article
Sprague-Dawley (SD), Fischer 344 (F344) and Lewis (LEW) rats are used in a wide variety of laboratory studies. Compared to SD and LEW rats, F344 rats show significantly greater activation of the hypothalamic-pituitary-adrenal (HPA) axis in response to acute stress, or to immunologic challenge. These differences in HPA axis responsivity have been the basis for numerous studies investigating strain differences in immunological and behavioral parameters. However, strain differences in the adaptation of the HPA axis response to prolonged stress, or to repeated stress, have not been investigated. This series of studies demonstrates that F344 rats maintain significantly higher ACTH and corticosterone levels than SD and LEW rats during a single prolonged stress session. Furthermore, F344 rats show virtually no habituation or adaptation of the corticosterone stress response during a single prolonged (4 h) stress session, or during stress sessions repeated over a period of 10 days. In contrast, SD and LEW rats show habituation both within and across stress sessions. Strain differences in HPA axis responsivity are also reflected in the significant adrenal hypertrophy observed in F344 rats (but not in SD or LEW rats) following repeated stress. These results show that strain differences in HPA axis responsivity, which are observed under conditions of acute stress, are further amplified during prolonged or repeated stress. These differences under prolonged or repeated stress conditions may consequently magnify the behavioral and immunological differences observed between strains under basal as well as challenged conditions.
The physiological role of the catecholamines (CA), adrenaline and noradrenaline in fish has been frequently reviewed, but the metabolic consequences of these hormones have received less attention. The purpose of this review is to examine the recent literature dealing with CA actions on whole fish and tissue metabolism. The CA increase glucose production both in vivo and in vitro, at least in isolated hepatocytes. Although the data are less clear, lipid mobilization is also a consequence of elevated circulating CA. The difficulty with using the whole fish for such studies is that CA may alter other circulating hormone levels, CA turnover in the circulation quickly, and it is difficult to define precisely the tissue being affected. Much of our understanding is derived, therefore, from the study of isolated tissues, and especially the hepatocyte. Catecholamines stimulate both glycogenolysis and gluconeogenesis in hepatocytes isolated from a large number of fish species. This review examines the steps involved in the signal transduction system, from the binding of CA to alpha- and beta-adrenoceptors to the ultimate effects of specific enzyme phosphorylation. Recent literature demonstrates that the complexity of the adrenoceptor system noted for mammals, also is expressed in fish. Adrenoceptor subtypes are specific to species, to tissues and to function of the tissues, and these issues are discussed especially as they are related to external and to internal stressors. Future research will pursue better definitions of the adrenoceptor systems, molecular biology of the components of these receptor systems and development of alternative cell models. There still remains a poor explanation of the reason for the diversity of adrenoceptor systems, and there are a number of fish systems that may provide unique opportunities to understand this question.
Article
Cells from goldfish and amphibian lymphoid organs, mainly leukocytes, express high affinity beta-adrenergic receptors specific for beta-adrenergic ligands (agonists: adrenaline, noradrenaline, terbutaline, and fenoterol; antagonists: CGP-12177, dihydroalprenolol, propranolol, atenolol, and butoxamine). The rank order of ligand potency does not allow their being classified into any known mammalian subtype. Among features that distinguish them from mammalian beta1 and beta2-adrenoceptors is much lower affinity for (-)-CGP-12177, obtained in both saturation and kinetic experiments (about 25 nM for goldfish head kidney cells). The density of receptors on goldfish and anuran cells is organ-dependent and comparable to that estimated on mammalian leukocytes. The extraordinarily high receptor density on salamander splenic cells (about 183,000) correlates with the large size of urodele cells. The competition experiments on goldfish cells with propranolol and CGP-12177 suggest the existence of yet another binding site, which may be either another beta-AR subtype, or a serotonergic receptor.
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In this review, James Downing and Jaleel Miyan outline emerging evidence for neural mechanisms that contribute to specific categories of host defence. Involvement of direct innervation in the adaptive control of immunological responses complements an established view of neuroendocrine-immune modulation. The challenge remains to understand the integrative and homeostatic functions of 'hardwiring' of peripheral immune effector sites, its bearing on disorder and potential for therapeutic modification.
Article
The effects of endosulfan, an organochlorine pesticide, on cortisol secretion in vitro were investigated in enzymatically dispersed head kidney cells of rainbow trout, Oncorhynchus mykiss. First, the conditions for maximal cortisol secretion were characterized by selecting the optimal concentrations of adrenocorticotropin (ACTH) (1 IU/ml) and N(6), 2'-o-dibutyryladenosine 3':5'-cyclic monophosphate (dbcAMP) (2 mM), incubation temperature (15 degrees ) and time period (60 min for ACTH, 120 min for dbcAMP), number of cells per incubation well (75 x 10(6)/ml), and osmolarity of the medium (270 mosmol/L). Exposure of head kidney cells to endosulfan decreased ACTH- or dbcAMP-stimulated cortisol secretion and cell viability in a concentration-dependent pattern and the doses required to disrupt cortisol secretion were significantly lower than doses lethal to the head kidney cells. The median effective concentration of endosulfan (EC(50), the dose that inhibits cortisol secretion by 50%) was 17.3 microM while the median lethal concentration (LC(50), the dose that kills 50% of the cells) was 308 microM. Our study identified endosulfan as an environmental endocrine disrupting chemical that interferes with the normal secretory function of teleost interrenal steroidogenic cells. Multiple sites may be affected within the steroidogenic cells since dbcAMP could not restore cortisol secretion in endosulfan exposed cells.
Article
A rainbow trout (Oncorhynchus mykiss) gene for tumor necrosis factor (TNF) has been cloned and sequenced. The cDNA contains an open reading frame of 738 nucleotides that translate into a 246 amino-acid putative peptide, with a 5' untranslated region (UTR) of 140 bp and a 3' UTR of 506 bp. Two potential N-linked glycosylation sites exist in the translation. The genomic sequence measures 2007 bp and contains three introns that intercept four coding exons. Expression studies using RT-PCR have shown that the trout TNF gene is constitutively expressed in the gill and kidney of unstimulated fish. Trout TNF expression could be up-regulated by stimulation of isolated head kidney leucocytes with lipopolysaccharide (LPS). Similarly, stimulation of a trout macrophage cell line (RTS11) with LPS resulted in an increased transcript level, as did incubation with recombinant trout interleukin (IL)-1 beta. The optimal timing for induction of TNF expression in trout macrophages was determined using recombinant trout IL-1 beta, where a clear induction was apparent by 2 h and peaked at 4 h. Evidence that this TNF gene is equivalent to mammalian TNF-alpha is discussed.
Article
Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT–PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.
Article
Immunological and cellular stress signals trigger the release of corticotropin-releasing hormone (CRH) from the spleen, thymus and inflamed tissue. In vivo and in vitro studies generally suggest that peripheral, immune CRH has pro-inflammatory effects and acts in a paracrine manner by binding to CRH-R1 and CRH-R2 receptors on neighboring immune cells. However, it now seems likely that some of the suggested pro-inflammatory actions of CRH may be attributed to novel CRH-like peptides or to the related peptide, urocortin, which is also present in immune cells and has especially high affinity for CRH-R2 receptors.
Article
Corticotropin-releasing hormone (CRH) is an important regulator of inflammation at the central level through hypothalamo-pituitary-adrenal (HPA) axis control of glucocorticoid secretion. Integrity of the HPA axis during autoimmune disease is critical in controlling the severity of inflammation, but the evidence for an HPA axis defect in the etiology of autoimmune diseases is not compelling. CRH secreted from leukocytes and neuronal terminals in peripheral tissues also plays a role in mediating inflammation. Elucidating the pathways underlying the expression of CRH, both central and peripheral, and interactions of CRH with other inflammatory mediators such as substance P, confers great potential for the development of a new generation of anti-inflammatory agents.