Review Article

Stressing the Inflammatory Network: Immuno-endocrine Responses to Allostatic Load in Fish

Ali Reza Khansari, Joan Carles Balasch, Felipe E. Reyes-López*, Lluís Tort*
Department of Cell Biology, Physiology and Immunology, Autonomous University of Barcelona 08193 Bellaterra, Spain
*Corresponding author:

Lluis Tort, Felipe E. Reyes-López, Department of Cell Biology, Physiology and Immunology, Autonomous University of Barcelona 08193 Bellaterra, Spain, E-mail:, Felipe.


Allostatic load, Fish, Inflammatory network, Regulatory systems

Fish and the other vertebrates subjected to challenges and stressors display a number of responses that involve many physiological compartments and mechanisms. Under these situations of an imposed allostatic load, the regulatory systems, i.e. neural, endocrine and immune exert a primary role in organizing the response. In this review, this role is revisited, in particular focusing in the head kidney, a unique structure with many physiological functions in fish. The repertoire of immune cells and molecules involved in the immune response is shown, paying particular attention to those that present an evolutive and functional difference compared to what is known in mammals. Overall, this review emphasizes the importance of the interaction between the regulatory systems, even more under pathogen challenges and stress situations.

Fish inhabit an intrinsically stressful medium. The physical and colligative properties of water facilitate the dispersion and resilience of xenobiotics, opportunistic pathogens and endocrine disruptors among other potentially distressing agents, hence translating the viral/bacterial load to a harmful allostatic load. Fish are considered a paraphyletic group of vertebrates, therefore numerous individual and populational differences exist in how fish species respond to stress circumstances, in particular on how individuals interpret and perceive the external signals or stimuli as a threat, thus activating or maintaining an active stress response. Imbalance in the systems generated by the allostatic load and the stress response has to be rapidly compensated because this situation may damage the host when it becomes chronic. Thus, a number of mediators from the neural, endocrine and immune systems will be activated to regulate the overall response to this allostatic load. Lesions or parasite occurrence in skin or gills, or local environmental changes in the gut caused by diet composition or pathogens, may also initiate local changes triggering the cytokines- or neural peptides-mediated immune response [1].

The physiological effects of waterborne fish diseases remain species-specific, but a common pattern emerges: in fish, the activation of Hypothalamic-Pituitary-Interrenal (HPI) and Brain-Sympathetic-Chromaffin cell (BSC) axis directs the neuroimmunoendocrine crosstalk [2], and the head kidney harbours the cellular scaffold that integrates the stress response. In the extensive systemic network of stress-activating immunitary defences and hormonal secretions, the proximal part of the kidney acts as a regulatory nodal organ, cradles the haematopoietic tissue

and releases the main stress hormones, corticosteroids and catecholamines (Figure 1). In fish, the head kidney encompasses the mammalian bone marrow and adrenal gland functionality in one single multifunctional structure (Figure 2).

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Figure 1.Stress axes in fish compared to mammals. HPI (Hypothalamus-Pituitary- Interrenal axis), BSC (Brain-Sympathetic-Chromaffin axis).

Cortisol (the main active corticosteroid in fish) is secreted by head kidney interrenal cells in a concatenated response after perception of stress by the CNS, involving release of hypothalamic corticotrophin releasing hormone (CRH) from hypothalamic paraventricular nucleus (PVN) and the adrenocorticotropic hormone (ACTH) by the pituitary gland. Released ACTH is recognized and bounded to melanocortin receptor 2 (MC2R) in the surface of interrenal cells and activates the steroidogenic signalling pathway leading to cortisol secretion as the final product of HPI axis activation [3]. The head kidney also receives sympathetic innervations, involving release of catecholamines (adrenaline and noradrenaline) by chromaffin cells.

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Figure 2.The stress response and the roles of the head kidney in fish.

The degree of activation of the neuroendocrine stress axis in fish depends on the type of stressors, intensity and also the duration of stimuli [4]. The stress response is characterized by its rapidity. Thus, the level of cortisol in zebrafish measured at frequent intervals over 1 hour, show initial increments already at 3 min post stress, peaking at 15 min [5].

Results obtained by our group also showed both in rainbow trout and sea bream subjected to acute stress the secretion of cortisol at the first minutes, peaking at 1 h, though the level can be still high after the first hours returning to control level at 12-16 h [6,7]. When the stressor is chronic or fish are subjected to a pathogenic stressor such as bath administration of Vibrio anguillarum, cortisol levels are maintained for several days or weeks after the initial exposure.

Both immune and endocrine cell types share common receptors [8]. Cortisol has receptors in different leukocyte cell types and it has been shown to modulate the immune response [9-11]. B- and T-cells are affected by cortisol reducing lymphocyte proliferation and reduction of natural killer cell (NK) activity and also impairment of antibody secretion [12]. These effects have been shown either in vitro or in vivo studies [13-15]. ACTH receptors have been detected in spleen and thymus and furthermore, teleost fish show corticotropic-releasing-factor (CRF) immunoreactivity in both gills and skin macrophage-like cells [16]. Other hormones such as leptins have also been found in the thymus and spleen of carp [17], indicating that the acute stress response may have a direct effect on immunological tissues [18]. Moreover, production of other hormones such as growth hormone and prolactin has been demonstrated to occur in fish immune cells [19].

The full account of the overall neuroimmunoendocrine interactions in fish are beyond the scope of this review. Here, we aim to briefly survey the main characteristics of fish immune system and describe the effects, characteristics and mediators of its stress-related endocrine regulation.

Besides the head kidney, the organography of immune tissues in fish resembles that of the mammals (Figure3), and, being vertebrates, both innate and adaptive branches of the immune system participate in defensive responses. The innate immunity is the primary and traditionally non-specific line of defence [21], and has been defined as the mechanism of recognition and response to pathogen-associated molecular patterns (PAMPs) such as polysaccharides, lipopolysaccharides (LPS), peptidoglycans, bacterial DNA or double strand viral RNA [21,22] Tissue damage also elicits a danger/damage-associated molecular patterns (DAMPs) inflammatory response when cellular contents or molecular immunity mediators are spilled in the extracellular matrix. The pathogenic signatures are recognized by pathogen recognition receptors (PRRs) found in immune cells, including lymphocytes, macrophages and neutrophils and soluble components on cellular compartments, endosomes, lysosomes and endolysosomes[23]. Currently, more than 17 Toll-like receptors (TLRs), a particularly widespread PRR, have been described in fish [24] with conserved TLR-signalling pathways [25], although with different features and high diversity [26].

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Figure 3.Overview of the immune response in fish. The figure depicts the immune organs, the mucosal-associated lymphoid tissues, and the main phases of the immune response. (For abbreviations, see the text).

The adaptive responses are orchestrated by the recognition of specific antigens by B and Tcell specialized receptors (BCR and TCR, respectively) that triggers a clonal expansion of selected lymphocyte populations and initiates the production of specific tailored antibodies. In fish, the levels of antibodies produced in response to an infectious process are maintained for a longer time compared to the mammal response model.

However, the dynamics of immune response is slower and smallerin magnitude [27]. Low temperatures may also have a higher impact on T-cell immune function (Figure 3), although this is a highly species-specific effect [28-30].

The immune defence in fish can be considered focused on innate parameters because the activation is quick, roughly constant and temperature independent [31], while the acquired immune response in fish takes at least couple of weeks to be fully active in comparison to innate immune response [32]. In zebrafish, the level of specific antibody was gently increased and reached highest levels within 28 days post vaccination [33]. It has been suggested that the ectothermy of fish may have limited the progression of their adaptive responses, favoring a more robust innate immunity [34]. Nevertheless, the adaptive immune response is of fundamental importance in fish defence since both T-cell responses and the synthesis of immunoglobulins are necessary to mount an efficient immune response against pathogens.

The aquatic environment harbours a wide variety of potential threats involving biological, physical and chemical agents that interact with the immune cells and molecules nested in the mucosal surfaces. According to mammalian nomenclature, these Mucosal-Associated-Lymphoid-Tissues (MALT) include (Figure 3) the gut-associated-lymphoid tissue (GALT), skin-associated-lymphoid-tissue (SALT), gills-associated-lymphoid-tissue (GIALT) and the most recently proposed nose-associated-lymphoid-tissue (NALT) [1,35,36].

The epithelium of these tissues is protected by a mucus layer which acts as a chemical and physical barrier, therefore becoming the immediate innate immune protection and acting as the first line of defence against pathogens [37]. In addition, mucosal tissues include a number of immune competent cells ready for an immediate response against immune threats. For instance, the gut mucosal immune system is armed by different cell-type populations including lymphocytes, plasma cells, granulocytes and macrophages both in the epithelium or dispersed in the lamina propia [38]. The GALT component of mucosal immunity can be traced back in the evolution of jawed vertebrates and it is considered to have played a significant role in forming the variegation of the molecules present in gut.

Skin is the largest mucosal surface in fish and it has been shown to be a metabolic, endocrine and immune active tissue [1]. There are some molecules that confer antimicrobial ability to the skin mucus such as lysozyme, complement components, lectins, proteolytic enzymes and Igs [39]. Thus, it has been reported that infection with Gyrodactylus derjavini induced the expression of TNF-α, TGF-β and COX2 in rainbow trout skin. Also husbandry stressors suppressed the activity of enzymes and genes involving cytokines such as IL-β1, TNF- α and IGF-1 in skin of Scophthalmus maximus after long -term stress [40]. Recently, it was reported that teleost skin evokes gut-like immune responses with a predominant role of IgT, a teleost immunoglobulin specialized in gut immunity [41].

We recently showed that acute handling stress and Vibrio anguillarum exposure promoted the expression of cytokines that are involved in inflammation response including IL-β1, TNF-α, IL-6, IL-10 in skin of rainbow trout and gilthead sea bream, therefore illustrating the importance of this barrier in inflammatory response. Together with skin, GIALT is the organ with most intimate contact with the aquatic environment, representing a crucial organ to deal with pathogens and also a portal of entrance for them. Thus, two common pathogens such as Vibrio anguillarum and Aeromonas salmonicida enter to fish via gills [42,43].

Most immune cell-types are present in gills including lymphocytes, macrophages, eosinophils, granulocytes, neutrophils and antibacterial proteins [44]. B-cells and the presence of IgT have been also reported in gills [45]. It has been described that bacterial challenges with Vibrio anguillarum and Aeromonas salmonicida induce changes in antibacterial proteins and cytokines expression in the gills of Atlantic cod (Gadus morhua) expressing IL-β1, IL-22, IFN-γ and IL-8. Therefore, it seems that the immune system implicated on fish mucosal barriers shows a rich repertoire of innate immune components and a clear interaction with the evironmental microbiota involved.

Finally, the mucosal immune system also relies on NALT. The olfactory sensory system appears to be one of the most ancient organs and its mucosa has been shown to be essential for all animals acting as mucosal defence against either waterborne antigens in aquatic vertebrates or airborne antigens in terrestrial vertebrates. Rainbow trout has been shown to be armed by NALT immune features showing innate and adaptive immune capabilities [46].

Trout olfactory tissue express immune features such as immune gene expression and immunoglobulin production, demonstrating IgT coating ability to the majority of bacteria [46]. As in mammals, the existence of nose mucosal immune features in fish indicates a vital role in facing with waterborne antigens.

A number of proteins are important players of the first-line inflammatory responses in fish, amongst them complement, lysozyme, and a plethora of antimicrobial proteins (AMPs). Upon antigen exposure, ARPs convey the cellular crosstalk and help to regulate the onset on innate immune responses.

Complement is composed of at least 35 proteins that are devoted to attacking the bacterial membrane. It can be initiated and activated by three distinct pathways partially overlapped: the classical pathway (CP), the alternative pathway (AP) and the lectin pathway (LP). The CP activation is induced by binding of antibody to the complement C1 complex, made by C1q and two serine

proteases (C1r and C1s), or by sticking directly of the C1q component to the bacteria surface. After hydrolysing of C3 to C3 (H2O), which obtains a C3b-like composition capable of binding to factor B(Bf) and factor D, leading in formation of the alternative C3 convertase [47]. The Alternative Pathway is initiated by the spontaneous hydrolysis of C3, an abundant protein of blood plasma that after successive reactions produce C3-convertase and after binding properdin, C5-convertase which binds sequentially a number of other complement proteins ending with a membrane attack complex that is able to lyse bacteria [48]. The Lectin Pathway is initiated by microbial polysaccharides that interact with circulating lectins. In fish, C3 component participates as a central complement component in all three pathways.

Therefore the presence of C3 even at early stages of the fish life has been shown to exist because of its pivotal role in innate immune system [49]. A study performed in our group illustrated that C3 is suppressed in liver as a producer of complement after cortisol administration in rainbow trout, whereas the repression was not induced in spleen, thus C3 appears to be regulated after stress conditions in tissue specific response [50]. One of the characteristics of the complement system in fish is the high variety of their proteins, thus offering a wide variety of isoforms, which contrasts with the lower variability found in mammals [51,52]. This variety of isoforms has been associated to a greater possibility of fish to respond to a wider variety of antigens at innate immune level [52].

Lysozyme is known as a bactericidal agent playing a crucial role in the innate immune system against broad spectrum of invading microorganisms. Although primarily lysozyme is concerned with defence against Gram-positive bacteria, also Gram-negative bacteria can be lysed by this enzyme [34]. Lysozyme can activate the complement system, therefore once the outer membrane of bacteria is destroyed by complement, and then lysozyme appears to be effective to process the pathogen [53]. Lysozyme is present in lymphoid tissue, plasma and body fluids and it has also been found on fish skin mucus [54,55].

Pentraxins (C-reactive protein, CRP and serum amyloid protein, SAP) are lectins, which are found in the body fluids of both vertebrates and in vertebrates and thus are involved in the so-called acute phase of response showing increments at serum level after tissue damage, trauma or infection. The pentraxins participate in the innate immune response through induction of complement proteins playing a role in the identification and elimination of apoptotic cells [56,57].

Fish can also fight pathogens by producing a broad spectrum of antimicrobial peptides (AMPs). Overall, AMPs are secreted into the circulation that is the major target for antigens. A long list of antimicrobial peptides in fish has been proposed with immunomodulatory properties [58].

Indeed, previous antecedents demonstrated that antimicrobial peptides are capable of modulating the cytokines response in mammals and fish [59,60] Also, in Coho salmon protection was shown against Vibrio anguillarum after a previous administration of AMPs [61].

Cytokines are secreted proteins with growth, differentiation, and activation functions that regulate the nature of immune responses [62]. Cytokines are active at the portals of entry resulting in elimination of the antigen. IL-1β is an endogenous pyrogen produced and released at the early stage response to infections, lesions and stress [63]. It has been reported the expression of IL-1β in fish following infection of both bacterial [64, 65] and also viral pathogens [66,67].

This is because in fish IL-1β is (as in mammals) one of the most important initiator of pro- inflammatory response with key roles on the stimulation of pro-inflammatory mediators such as other cytokines and prostaglandins in macrophages, and activation of lymphocytes [68,69]. IL-1β can activate the expression of IL-6, a pleiotropic cytokine which has pro-inflammatory function, being induced by LPS, poly I:C and IL-1β both in the macrophage cell line RTS-11 and head kidney macrophages [70]. IL-6 also promotes the differentiation of B-cells into plasma cells and activating cytotoxic T cells [71,72]. It has been also reported that IL-6 can significantly down-regulate the expression of pro-inflammatory cytokines, suggesting a potential role of trout IL-6 in limiting host damage during inflammation [71].

Other relevant pro-inflammatory cytokine is TNF-α, which plays an important role in a wide range of host responses, including cell proliferation and differentiation but also necrosis and apoptosis. This cytokine can stimulate the acute phase response and is one of the first to be released in response to pathogens as well as after stress episodes [73]. Also, TNF-α is able to exert its effect in many organs as well as induction of IL-6 together with IL-β [74]. Beyond the systemic immune organs which are directly implicated in immune response, the expression of IL-β1 and TNF-α has been also observed in the brain of sea bream and sea bass after Nodavirus exposure [75] indicating that other organs may be involved in the cytokine synthesis and, therefore, in the fish response against pathogens. The pro-inflammatory cascade is strictly regulated by anti-inflammatory mediators, TGFβ1 and IL- 10, responsible to maintain the response under control to avoid tissue damage [76,77]. TGFβ1 is often associated with IL-10 as an immunosuppressive cytokine [76] regulating the immune response by blocking the activation of lymphocytes and monotype derived phagocytes and promoting tissue repair after a local inflammatory response [77].

Importantly, the expression of TGFβ1 has also been associated to fish disease resistance, thus promoting a persistence phenotype in those fish infected with viral pathogens [66,79]. On the other hand, IL-10 is produced by activated macrophages, B cells and T cells [80], thus playing a pivotal role on the resolution of infection and, therefore, in reducing tissue damage caused by inflammation [81,82]. Taking together, the cytokine response is crucial for fish to induce an efficient and tightly regulated immune response.

Immunoglobulins are produced by B-cells [1]. Immunoglobulins can be categorized into different classes according to the nature of the C domain of the heavy (H) chain. Until recently fish were thought possessing only IgM and IgD, but interestingly in 2005 new immunoglobulins heavy chain IgT and IgZ were reported in rainbow trout and zebrafish, respectively [83,84]. IgT is chiefly polymeric and it is expressed in the gut mucus in much higher numbers than that observed in the serum, while IgM appears to be circulating in the serum.

Although production of specific antibodies against pathogens is assumed to be one of the efficient routes to cause immunoprotection, there has been suggested that because of low affinity of IgM to pathogen, antibody induction cannot prevent pathogen infection at early stages [85]. Therefore, IgT is proposed to play an essential role in the mucosal immune response [86]. Unpublished results of our group also show that the transcription of IgM can also be regulated after acute handling stress, though a previous study showed immunoglobulin M expression after temperature changes in gills, spleen, head kidney and intestine [87]. Also it has been shown that different environmental factors such as salinity influenced on the circulating level of immunoglobulins [88].

The innate immune response is also characterized by a pivotal action of macrophages as one of the primitive players of the host response to tissue invasion, resulting in phagocytosis and pathogen killing. A study performed by Mosser and colleagues [89] suggested that distinct pathogen invasions usually determine macrophages activation patterns into classically activated or alternatively activated macrophages. The activation of these macrophages normally result in secretion of pro-inflammatory cytokines such as IL-β1, TNF-α, IL-12 and IL-23, and also chemokines such as CXCL8-11. Thus, they take part in host defence against intracellular pathogens [89]. Alternatively activated macrophages are also known as an anti-inflammatory mediators playing essential role in protection of the host. After presence of pathogen in the host and activation of macrophages, the triggered response has to be rapidly controlled through phagocytosis, tissue repair and resolution of the infection. These macrophages regulate the pro-inflammatory response by imposing the anti-inflammatory activities to suppress IL-12 and boosting the production of IL-10 to protect the host form damage [89].

The identification of T cell markers such as α and β T cell receptor genes (TCR), CD3, CD4, CD8, CD28, CD40L, and also the presence of cytokines and chemokines suggest the presence of T helper (Th)1, Th2, Th17 and subsets of regulatory T cells in teleosts [62]. Lymphocytes express three distinct functions including provision of appropriate microenvironment for development of immune effector cells, mediating quality control (positive and negative selection) and regulating memory and pathogen elimination through the synthesis of immunoglobulins [90]. An important feature of fish lymphocytes that has been poorly studied is the correlation between nervous and immune system. Most of the immune components, especially lymphocytes, express receptors for neuromediators, thus being another bilateral regulatory network through direct neuro- immune connections. In vertebrates, CD4 T lymphocytes recognize peptides presented by MHC class

II molecules on antigen-presenting cells and proliferate in response to autocrine IL-2. The presence of CD8 (+) dendritic-like cells provides evidence of the presence of specialized antigen-presenting cells [91]. In fish, functional activities related to Th1 and Th2 response have been shown in rainbow trout and zebrafish observing expression of CD4 [92,93]. Contrary to tetrapods, teleosts contain two CD4 genes, called CD4-1 and CD4-2 [74,95]. The biotechnological advances have allowed the generation of specific antibodies anti-CD4 in salmonids. Thus, it has been described the existence of T lymphocytes CD4-1(+) CD3(+) [96] but also lymphocytes CD4-1(+)CD4-2(+) and CD4-2(+) and, in addition, one CD4-1(+) myeloid subset [97]. Thus, the utilization of antibodies will provide additional evidence to understand the role of different cell-type populations on the immune response in teleost fish.

Among the most striking features of lymphocytes of fish is their ability to perform phagocytosis, as compared to mammalian lymphocytes. Three major B cell lineages have been described in teleosts, those expressing either IgT or IgD, and the most common lineage which co-expresses IgD and IgM. Thus Li et al. demonstrated for the first time that about 40% of fish lymphocytes have phagocytic activity and intracellular bactericidal capacities. This finding represented a paradigm shift as professional phagocytosis was believed to be exclusively performed by some cells of the myeloid lineage This property of fish B cells suggest that lymphocytes have evolved to maintain early functional capacities of cells that have been lost further in the evolution as both white and red blood cells become more specialized. This involves a less specialization but higher versatility of fish blood cells to fight against pathogens. This phagocytic capacity was also found as well in other groups of vertebrates, amphibians and reptiles, suggesting that this innate capacity was evolutionarily conserved in certain B cell subsets of vertebrates. Moreover, it appears that phagocytic B- 1 B cells have a potent ability to present particulate antigen to CD4(+) T cells [98]. At present, these studies that were carried out originally on fish B cells have led to the discovery of new innate and adaptive roles of B cells in mammals [98].

Erythrocytes are the most abundant cell in the circulatory system. Fish erythrocytes half-life has been shown to be 80-500 days compared to 120 days in humans[99].

Nevertheless, the main differential characteristic is that fish red blood cells present nucleus, as in other lower vertebrates, contrary to mammals. It is assumed that during the evolution fish erythrocytes conserved the nucleus and therefore the ability to transcript, translate and produce proteins, whereas mammalian erythrocytes are not able to do it because of the lack of nucleus and they evolved in a deeper specialization on oxygen carrying. Because fish erythrocytes express proteins it has been investigated whether these cells could also have a role in the immune system and therefore express specific immune-related proteins (receptors, effectors). Morera and Mackenzie

[100] demonstrated the existence of such direct role of erythrocytes in the immune response and thus it was demonstrated the presence of TLR receptors and a positive response to viral and bacterial molecular patterns [100]. Therefore, although the immune role of erythrocytes may be moderated or low, they show the potential of immune reactivity and therefore the advantage to be the most abundant cell-type in bloodstream. Although fish are protected with innate mechanisms and mucosal barriers as a first line of defence, once pathogens clear this first line and access to circulation, the erythrocytes could play a role in the circulatory bed. It has also been described that erythrocytes take part in facilitating the clearance of pathogen by macrophages [101], providing more evidence in the role of nucleated erythrocytes as an immune-like cell type.

Not only erythrocytes have been shown to be involved in immune defence, but also thrombocytes have been reported to play a putative immune role. They can present round, oval, ellipsoidal or spiked forms [102] and their main function is a regulator of hemostasis and blood clotting [103] but also it has been evident that beyond hemostasis, they participate in modulating the innate and adaptive immune response initiating the inflammatory response through interaction with leukocytes in case of injury [104,105]. The expression of TLRs on platelets has also been found on birds, mice and human [106], but the mechanisms behind the immune role of thrombocytes have not yet been elucidated.

After sensing a stressor (physical, chemical, environmental) the fish responds to stress activating the CNS to release stress hormones which will be the responsible to promote the physiological mechanisms able to counteract these stimuli [107]. The classical view of these stress hormones is that cortisol, adrenaline and ACTH are suppressors of the immune system, but recent work demonstrates that hormones are modulating rather than suppressing agents [4].

Although in mammals and fish adrenaline has been found to suppress the expression of some inflammatory cytokines as well as inhibiting the phagocytic activity of leukocytes and also decreasing plasma levels of IgM [108,109], an enhancing effect of adrenaline was reported in mammals showing up-regulation of IL-6 [110]. In a recent study, we also observed the enhancing influence of adrenaline on IL-β and IL-6 in sea bream head kidney. Thus, the immunomodulatory influence of adrenaline on immune system remains to be investigated.

The secretion of cytokines after pathogen recognition by the host to mount an efficient response will also induce a regulatory effect of these cytokines on HPI axis. The pro-inflammatory cytokines IL-1, TNF and IL-6 induce glucocorticoid and ACTH secretion following infection and inflammation [111]. In the same way, recent results from our lab show that in rainbow trout and gilthead sea bream HPI axis is simulated to release cortisol after the exposure to Vibrio anguillarum bacterin (not published data).

Also, it has been reported that the stimulation with PAMPs, such as LPS, elicits a rapid and dose- dependent activation of HPI which results in the release of ACTH and cortisol [112]. Therefore, the direct influence of immune system mediators, such as cytokines, could also lead to hormone secretion into bloodstream. However, the detailed mechanism by which the fish HPI axis is stimulated by cytokines has not been fully described to date.

In addition to immune cells, non -immune cells are able to secrete cytokines. Thus, a large number of cytokines are influencing the HPI axis such as IL-β1, TNF- α and IL-6 [113]. Regarding the nervous system, neurons of the hypothalamus and pituitary such are capable of producing cytokine such as TGF, IL10 and IL-18 [114].

Glucocorticoids (GCs) are known to modulate the immune system in vertebrates exerting a complex action on a number of immune components [115].

Our group showed the effect of cortisol on induced-gene expression profile with LPS in rainbow trout macrophages. Thus, cortisol inhibits the expression of genes involved in cell adhesion, cell surface receptor linked signal transduction, humoral immune responses, apoptotic regulation and complement system [116]. In fact, it has been evident that roughly every cell type of the immune system is affected by glucocorticoids [117]. There is less studies in which cortisol was not able to modulate gene transcription. In rainbow trout macrophages Castro et al. [94] did not induce changes after different co-incubation times with cortisol and immune stimuli administration. Previously, both the suppressing and enhancing (immunomodulatory) effects of cortisol have been largely reported in fish hepatocytes and head kidney cells [14,15,23]. The cortisol administration has been shown to suppress the immune response at cytokines level for IL-β1, IL-6, TNF-α and TGF-β1 in sea bream but not in rainbow trout. Therefore, this suggests that GCs effect in fish could act in a tissue-specific and species-specific manner.

Catecholamines have not been studied as much as glucocorticoids in fish because of sampling difficulties related to the fact that catecholamines are released in seconds, therefore difficulting the obtention of basal values

[119]. In addition, a simple elevation of adrenaline in blood contributes to impairing the regulation of blood Na+ and Cl-, thereby modifying the ion concentration balance in plasma and causing osmotic disturbances. Physiological and pharmacological concentrations of adrenaline have been shown to increase HSP70 in hepatocytes of salmonid

[119]. There is evidence that suggests an effect of adrenaline also in the modulation of the immune response. Thus, previous studies have shown that nitric oxide (NO), TNF-α and IL-10 were simultaneously increased following LPS treatment in vitro and that adrenaline dampened both NO and TNF-α with concurrent enhancement of IL-10 [109]. Also, Kepka and colleagues [120] have shown that injection of zymosan plus adrenaline reduced the percentage of monocytes/macrophages 24 h after injection. Also, a significant increase of apoptosis and decrease of IL-β1 was observed in the monotype/macrophages treated with zymosan and adrenalin, while at 24 h IL-β1 was increased by adrenaline [120].

The role of ACTH on immune system has been poorly investigated in fish, though its implication in cortisol secretion pathway. ACTH was shown to be one of the first neuropeptides binding to immune component cells thus modulating the immune response in mammals, and also ACTH induced an increased cytotoxic response in T-lymphocytes [121]. In fact numerous studies have illustrated immunological influence of ACTH suppressing the activation of both human granulocytes and invertebrates immunocytes [122]. ACTH has been found to prevent antibody response to T-cells as well as repressing interferon gamma secretion (IFN-γ) in murine splenocytes [123]. ACTH can also promote B-lymphocytes growth and differentiation, modulating the IgM secretion and proliferation of phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells [124]. Thereby the immune modulatory effect of ACTH after neuroendocrine activation can be also expected in fish. The presence of ACTH was detected by immunocytochemistry in all stages of Dicentrarchus labrax even 2 days post hatch, revealing that the MC2R receptor should be present at early stages [18,125]. Immunoreactivity of ACTH has also been shown, suggesting an important role of this neuropeptide to preserve the alteration of body haemostasis within the first fish life developmental stage [18]. Moreover, it has also been shown that ACTH can directly regulate catecholamine secretion in rainbow trout [126], thus it could be involved in the endocrine-immune interaction through both stress axes.

Aside from ACTH secretion by neuroendocrine system, ACTH concentration was also detected in immune cells presenting in mammalian white blood cells and invertebrate immune components that have an essential role in phagocytosis. In fact, a study performed by Csaba and Pallinger illustrated the autoimmune regulation of the ACTH in immune cells [127]. In other words, immune cells can be well innervated and express receptors for neurotransmitters and neurohormones.

It is noteworthy that the interpretation of the in vivo obtained results can be a challenge fundamentally because the interaction between different effectors of the endocrine and nervous systems and their differential influence on the immune response. Thus, the in vitro model can be useful to study the individual impact of a specific hormone on immune cells or pathways. Previous studies performed in our group have revealed the stimulatory effect of ACTH on cytokines expression, enhancing TNF-α, IL-6 and TGF-β expression after 1 h of incubation. However, ACTH has also an immunosuppressive effect on IL-β1, IL-6, TNF-α, IL-10 and TGF-β1 after 2 h of exposure in sea bream head kidney [13]. On the other hand, ACTH was not able to mediate any modulatory effect in sea bream after stimulation with LPS and Vibrio anguillarum in either sea bream or rainbow trout [13]. Therefore, hormones would play a modulatory rather than suppressive role on the immune system, but the specific actions resulting from these interactions between systems are subjected to the specific experimental conditions such as the previous history of immune and endocrine inputs.

Overall, during activation of the immune system or after an infectious process, glucocorticoids function to modify inflammatory response which is normally considered as an energy saving mechanism during stress incidents in which more energy resources are required and so, an additional allostatic load [128]. Thereby, not only is immune activity dampening, but immune cells may be catabolized as a source for protein and glucose, hence the function of GCs and in particular the bidirectional interaction between neuroendocrine and immune system appear to be indispensable as well as crucial for the animal integrity and thus cope with the pathogens invasion.

As the communication between neuroendocrine and immune systems is bidirectional, endocrine activation also appears to be mediated by the immune system. In fact, the challenge involved in the process of an infection results in an allostatic load for the animal, therefore triggering a stress response resulting in the release of stress hormones. Thus, the presence of the pathogen Lepeophtheirus salmonis in Atlantic salmon increased the level of cortisol as well as glucose [129]. A sea louse, one of the major pathogens for the farmed Atlantic salmon, induces cortisol and glucose elevations [130]. An acute cortisol response was also observed in channel catfish following an E. ictaluri challenge [131], similar than that observed in rainbow trout, Oncorhynchus mykiss, following Vibrio anguillarum infection [132]. The injection of Pseudomonas anguilliseptica, an opportunistic pathogen for sea bram, induced an increase of cortisol, but only after 3 days of injection [134]. Regarding the infection consequences, it has been shown that under infection and inflammation, expression of IL-1, TNF and IL-6 induced secretion of glucocorticoids and ACTH [111,114]. Experiments using bacterial lipopolysaccharides (LPS) also showed induction of the HPI axis response. Van Enckevort et al., treated tilapia with Escherichia coli LPS stimulated the HPI axis and CRH and cortisol release but produced inhibition of ACTH and stimulation of α- MSH release after in vitro pituitary treatment [134-142], although there was no effect of LPS on plasma cortisol in channel catfish [143].

In yellow perch, Haukenes and Barton showed that an injection of LPS (3 mg/kg) elicited an increase of cortisol secretion even stronger than under handling stress (Haukenes and Barton, 2004) . In Oncorhynchus mykiss injections of trout recombinant interleukin-1 beta (rIL-1 beta) or E. coli lipopolysaccharide (LPS), at concentrations known to induce immune/inflammatory responses in vivo (0.1-0.6 nmol/kg and 1.3 mg/kg respectively), significantly elevated plasma cortisol levels in a dose - and/or time-dependent manner [144]. Other studies also showed increase of cortisol and cortisol receptor levels (GR) in sea bream, although at much higher concentrations than for mammals [133] which may indicate a higher tolerance of fish to microbial PAMPs. LPS treatment on head kidney primary culture also showed an increase of the expression of the StAR protein, the key step to activate the cortisol production in fish [145,6]. Moreover, recent results from our laboratory showed that inactivated Vibrio anguillarum affected the HPI axis in rainbow trout and gilthead sea bream by releasing cortisol together with the up-regulation of cytokines including IL-β1, TNF-α and IL-6. Altogether, these antecedents indicate that the PAMP-mediated PRR identification and activation of the immune response may also activate the neuroendocrine machinery interacting each other to mount an efficient global physiological.

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Citation: Khansari AR, Balasch JC, Reyes-López FE, Tort L (2017) Insights into Genomic Variation within Salmonella enterica. J Mar Sci Res Technol 1:002.

Published: 24 July 2017

Reviewed By : Dr. Muhammet Turkoglu,


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