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In both mammals and fish, the circadian system is composed of oscillators that function at the cellular, tissue, and system levels and show the cyclic expression of clock genes. The organization and functioning of the biological clock in fish has not yet been characterized in detail, therefore, in the present study, an extensive analysis of the rhythmic expression of the main components of the biological clock in the central and peripheral organs of common carp was performed. The diurnal changes in clock gene expression were determined with respect to the subjective light cycle in fish exposed to constant light or darkness. It was found that the pattern of expression of clock, bmal, per and cry genes in carp was highest in the brain, pituitary gland, and retina. The peak clock and bmal expression was phase aligned with the lights off, whereas both per genes show similar phasing with acrophase close to light onset. The expression of cry genes varied depending on the type of tissue and the subtype of gene. The diurnal changes in the expression of clock genes demonstrates that, in particular, the expression of the clock in the retina shows endogenous oscillations independent of the influence of light. The data suggest that in carp, the time-varying expression of individual genes allows for a diverse and tissue-specific response to secure oscillations with variable phase and period.

Mechanizm reakcji na stres jest konserwatywny ewolucyjnie, w związku z tym mimo odmiennego środowiska życia, odpowiedź stresową ryb doskonałokostnych charakteryzuje wiele podobieństw do kręgowców lądowych. Obejmują one główne przekaźniki osi układ współczulny-komórki chromafinowe (odpowiednik osi układ współczulny-rdzeń nadnerczy) oraz osi podwzgórze-przysadka-komórki śródnerkowe nerki głowowej (funkcjonalny analog osi podwzgórze-przysadka-nadnercza), a także ich główne funkcje. Współdziałanie układu neuroendokrynnego i układu odpornościowego stanowi integralną część fizjologii, nieodzowną dla utrzymania homeostazy. Zakłócenie złożonych interakcji neuro-endokrynno-immunologicznych ma negatywny wpływ na stan zdrowia i ogólny dobrostan zwierząt i ludzi. Ryby doskonałokostne stanowią szczególnie intrygujący model w zakresie badań wpływu stresu na odporność, ponieważ funkcje immunologiczne i hormonalne są u nich zintegrowane w obrębie jednego narządu - nerki głowowej. W konsekwencji procesy immunologiczne podlegają bezpośredniej modulacji parakrynnej. Celem niniejszej pracy jest przedstawienie przeglądu aktualnej wiedzy na temat przebiegu reakcji stresowej u ryb i jej roli w regulacji odporności ryb.

Tilapia lake virus (TiLV) is a novel enveloped orthomyxo-like virus with a genome of 10 segments of linear negative-sense single-stranded RNA. It causes massive mortality of wild and farmed tilapia species and because of its spread in Asia, Africa, South and North America, it is considered a threat to tilapia aquaculture. Here, we have evaluated the possible use of zebrafish (Danio rerio) to study immune response and host-pathogen interactions during an infection with TiLV. Adult zebrafish were infected with TiLV by intraperitoneal (i.p) injection or by cohabitation. Increased viral load was observed in liver, spleen and kidney of i.p. injected fish at 1, 3, 6, and 14 days post infection (dpi) but not in fish from the cohabitation group (only liver was tested). We also demonstrated that in spleen and kidney i.p. injection of TiLV induced up-regulation of the expression of the immune-related genes encoding pathogen recognition receptors involved in sensing of viral dsRNA (rig-I, tlr3, tlr22), transcription factors (irf3, irf7), type I interferon (infϕ1), antiviral protein (mxa), pro-inflammatory (il-1β, tnf-α, il-8, ifnγ1-2) and anti-inflammatory (il-10) cytokines, CD4 markers (cd4-1, cd4-2), and IgM (igm). Moreover, tissue tropism of TiLV and histopathological changes were analyzed in selected organs of i.p. injected zebrafish. Our results indicate that zebrafish is a good model to study mechanisms of the TiLV infection and to follow antiviral responses.

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

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

Arginase is the manganese metalloenzyme catalyzing the conversion of l-arginine to l-ornithine and urea. In vertebrates, arginase is involved in the immune response, tissue regeneration, and wound healing and is an important marker of alternative anti-inflammatory polarization of macrophages. In invertebrates, data concerning the role of arginase in these processes are very limited. Therefore, in the present study, we focused on the changes in arginase activity in the coelomocytes of Eisenia andrei. We studied the effects of lipopolysaccharide (LPS), hydrogen peroxide (H2O2), heavy metals ions (e.g., Mn2+), parasite infection, wound healing, and short-term fasting (5 days) on arginase activity. For the first time in earthworms, we described arginase activity in the coelomocytes and found that it can be up-regulated upon in vitro stimulation with LPS and H2O2 and in the presence of Mn2+ ions. Moreover, arginase activity was also up-regulated in animals in vivo infected with nematodes or experiencing segment amputation, but not in fasting earthworms. Furthermore, we confirmed that the activity of coelomocyte arginase can be suppressed by l-norvaline. Our studies strongly suggest that similarly to the vertebrates, also in the earthworms, coelomocyte arginase is an important element of the immune response and wound healing processes.

Skin regeneration requires a three-dimensional (3D) scaffold for cell adhesion, growth and proliferation. A type of the scaffold offering a 3D structure is a nonwoven material produced via a melt-blown technique. Process parameters of this technique can be adapted to improve the cellular response. Polylactic acid (PLA) was used to produce a nonwoven scaffold by a melt-blown technique. The key process parameters, i.e., the head and air temperature, were changed in the range from 180–270 °C to obtain eight different materials (MB1–MB8). The relationships between the process parameters, morphology, porosity, thermal properties and the cellular response were explored in this study. The mean fiber diameters ranged from 3 to 120 µm. The average material roughness values were between 47 and 160 µm, whereas the pore diameters ranged from 5 to 400 µm. The calorimetry thermograms revealed a correlation between the temperature parameters and crystallization. The response of keratinocytes and macrophages exhibited a higher cell viability on thicker fibers. The cell-scaffold interaction was observed via SEM after 7 days. This result proved that the features of melt-blown nonwoven scaffolds depended on the processing parameters, such as head temperature and air temperature. Thanks to examinations, the most suitable scaffolds for skin tissue regeneration were selected.

In carp aquaculture, hormonal manipulation with an analog of GnRH (Ovopel) and carp pituitary extract (CPE), which act at different levels of the hypothalamic-pituitary-gonadal axis, is a routine practice to enhance sperm production. Our recent studies revealed that hormonal stimulation of male carp was associated with changes in the seminal plasma proteome, including blood origin proteins. Here, we explored whether Ovopel and CPE could affect the blood proteome of male carp. Both preparations induced increases in semen volume, total number of sperm, and testosterone level. However, hormonal stimulation did not affect the plasma cortisol and glucose levels. A comparative proteomic analysis of carp blood plasma between the control (PBS) and the hormonally treated males revealed significant changes (>1.2

Gyrodactylids are ubiquitous ectoparasites of teleost fish, but our understanding of the host immune response against them is fragmentary. Here, we used RNA‐Seq to investigate genes involved in the primary response to infection with Gyrodactylus bullatarudis on the skin of guppies, Poecilia reticulata, an important evolutionary model, but also one of the most common fish in the global ornamental trade. Analysis of differentially expressed genes identified several immune‐related categories, including IL‐17 signalling pathway and Th17 cell differentiation, cytokine‐cytokine receptor interaction, chemokine signalling pathway, NOD‐like receptor signalling pathway, natural killer cell‐mediated cytotoxicity and pathways involved in antigen recognition, processing and presentation. Components of both the innate and the adaptive immune responses play a role in response to gyrodactylid infection. Genes involved in IL‐17/Th17 response were particularly enriched among differentially expressed genes, suggesting a significant role for this pathway in fish responses to ectoparasites. Our results revealed a sizable list of genes potentially involved in the teleost‐gyrodactylid immune response.

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

Purpose: The polymeric porous surface of fibres (PLA) may influence the kinetics of release of biologically active compounds (gentamicin, G and ethacridine lactate, R) affecting development of a bacterial biofilm. Methods: The porous fibres with different morphology were manufactured by the electrospinning method from ternary systems composed of PLA and selected solvents. Fibres morphology was examined using a scanning electron microscopy (SEM), their structure was analyzed by FT-IR ATR spectroscopy and differential scanning calorimetry (DSC). Changes in the drug release profile were measured using ICP/UV-Vis methods and the resulting bactericidal or bacteriostatic properties were tested by two-layer disk diffusion test in relation to various drug incorporation methods. Results: The porous fibres can be applied to produce drug-bearing membranes. The spectroscopic studies confirmed incorporation of gentamicin into the fibres and the presence of ethacridine lactate on their surface. Bimodal fibres distribution (P3) promoted faster release of gentamicin and ethacridine lactate from P3G and P3R materials. The electrospinning process coupled with the vapor induced phase separation influenced the glass transition temperature of the porous polymer fibres. The pre/post-electrospinning modification influenced the glass transition, maximum temperature of cold crystallization and melting point of the porous membrane, compared to the neat polymer. The polylactide fibres with gentamicin showed strong bactericidal effect on Gram-positive bacteria, while fibres with ethacridine lactate were bacteriostatic. Conclusions: The obtained fibres with complex surface morphology can be used as a membrane in active dressings as they make it possible to control the release profile of the active compounds.

International audience; Tilapia lake virus (TiLV; genus: Tilapinevirus, family: Amnoonviridae) is a recently characterised enveloped virus with a linear, negative-sense single-stranded RNA genome, which causes high mortality in tilapia species. In the present study, we demonstrated that zebrafish (Danio rerio) larvae are susceptible to TiLV infection upon systemic injection. TiLV replicated in zebrafish larvae and caused their high mortality (of about 70%). Histopathological examination revealed that TiLV infection caused pathological abnormalities in zebrafish larvae that were well visible within the brain. Moreover, gene expression analysis revealed that TiLV infection induced up-regulation of the expression of the immune-related genes encoding pathogen recognition receptors involved in sensing of viral dsRNA (rig-I (ddx58), tlr3, tlr22), transcription factors (irf3, irf7), type I interferon (infϕ1), antiviral protein (mxa), and pro-inflammatory cytokine (il-1β). We also demonstrated the protective role of the recombinant zebrafish IFNϕ1 on the survival of zebrafish larvae during TiLV infection. Our results show the importance of type I IFN response during TiLV infection in zebrafish larvae and demonstrate that zebrafish is a good model organism to study interactions between TiLV - a newly emerging in aquaculture virus, and fish host.

In mammals, several non-RLR DExD/H-box RNA helicases are involve in sensing of viral nucleic acids and activation of antiviral immune response, however their role in the immune defense of fish is much less known. In this study, the expression profile of non-RLR DExD/H-box RNA helicase genes: ddx1, ddx3, dhx9, ddx21 and dhx36, was studied in zebrafish (Danio rerio) and common carp (Cyprinus carpio L.) during infection with two RNA viruses: spring viremia of carp virus (SVCV) and Chum salmon reovirus (CSV). Bioinformatic analysis of the amino acid sequences of the core helicase of DDX1, DDX3, DHX9, DDX21 and DHX36 in zebrafish and common carp revealed presence of all conserved motifs found amongst all other species, with the exception of common carp DHX9 which do not possess motif V. The transcripts of studied DExD/H-box RNA helicases were found in zebrafish ZF4 cell line as well as in all studied organs from zebrafish and common carp. The expression study demonstrated the up-regulation of the expression of selected non-RLR DExD/H-box RNA helicases during viral infections in ZF4 cell line (in vitro study) and in zebrafish and common carp organs (in vivo study). DDX1 was the only DExD/H-box RNA helicase which expression was repetitively up-regulated during in vivo infections with SVCV and CSV in zebrafish and SVCV in common carp. In ZF4 cells and kidney of common carp, viral infection-induced up-regulation of DExD/H-box RNA helicases preceded the up-regulation of type I IFN gene. Our results suggest that studied non-RLR DExD/H-box RNA helicases might be involved in antiviral immune response in fish.

Inflammation is the evolutionary conserved immune response to harmful stimuli such as pathogens or damaged cells. This multistep process acts by removing injurious stimuli and initiating the healing process. Therefore, it must be tightly regulated by cytokines, chemokines, and enzymes, as well as neuroendocrine mediators. In the present work, we studied the immunoregulatory properties of 17β-estradiol (E2) in common carp. We determined the in vitro effects of E2 on the activity/polarization of macrophages and the in vivo effects during Aeromonas salmonicida-induced inflammation. In vitro, E2 reduced the lipopolysaccharide (LPS)-stimulated expression of pro- and anti-inflammatory mediator genes but did not change the gene expression of the estrogen receptors and of aromatase CYP19. In contrast, in vivo in the head kidney of A. salmonicida-infected fish, E2-treated feeding induced an upregulation of gene expression of pro-inflammatory (il-12p35 and cxcb2) and anti-inflammatory (arginase 1, arginase 2, il-10, and mmp9) mediators. Moreover, in infected fish fed with E2-treated food, a higher gene expression of the estrogen receptors and of the aromatase CYP19 was found. Our results demonstrate that estrogens can modulate the carp innate immune response, though the in vitro and in vivo effects of this hormone are contrasting. This implies that estradiol not only induces a direct effect on macrophages but rather exerts immunomodulatory actions through indirect mechanisms involving other cellular targets.

Both in mammals and in fish, CXC chemokines activate leukocytes and regulate their migration both under normal physiological and inflammatory conditions. Moreover, in mammalian neutrophils CXC chemokines also stimulate the formation of neutrophil extracellular traps (NETs). Here, we investigated the effects of recombinant carp CXCL8s and CXCb1 on NET formation in neutrophils from the head (HK) and trunk (TK) kidney of carp. We found that neither recombinant CXCL8s nor CXCb1 stimulated DNA release in HK-derived neutrophils, while in TK-derived cells rcCXCb1 stimulated the release of NETs, composed of extracellular DNA co-localized with citrulline H3 histone and neutrophil elastase. Furthermore, CXCb1-induced NET release required NADPH oxidase activity, while it did not change upon treatment with CXCR inhibitors. In conclusion, we demonstrated, for the first time in fish, that CXCb1 chemokine induces formation of NETs in TK-derived neutrophils and this process is ROS-dependent. The difference between HK and TK-derived neutrophils is probably related to differences in the maturation state of these cells.

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

Pro-inflammatory cytokines (IL-1b, IL-6, TNFa) and prostagandins (e.g. PGE2) produced during inflammation regulate the activity of leukocytes and stimulate the production of acute phase proteins, e.g. CRP. They also affect various brain structures and induce a series of behavioral changes collectively described as sickness behavior. It consists of, among others, fever, appetite disorders, locomotor and cognitive activities as well as disorders of sleep structure and length. Most often, their action shortens the falling asleep phase and paradoxical sleep (REM) phase, and extends the slow-wave sleep phase (SWS). Similar effects are also induced by IL-2 and cytokines belonging to the interferon family, including the most important for the antiviral response - type I interferons (IFN I). The direction and severity of these changes depend on the phase and severity of the infection. Behavioral changes induced by pro-inflammatory cytokines are highly adaptive and evolutionarily conserved.

Recognition of the non-self signature of invading pathogens is a crucial step for the initiation of the innate immune mechanisms of the host. The host response to viral and bacterial infection involves sets of pattern recognition receptors (PRRs), which bind evolutionarily conserved pathogen structures, known as pathogen-associated molecular patterns (PAMPs). Recent advances in the identification of different types of PRRs in teleost fish revealed a number of cytosolic sensors for recognition of viral and bacterial nucleic acids. These are DExD/H-box RNA helicases including a group of well-characterized retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) and non-RLR DExD/H-box RNA helicases (e.g., DDX1, DDX3, DHX9, DDX21, DHX36 and DDX41) both involved in recognition of viral RNAs. Another group of PRRs includes cytosolic DNA sensors (CDSs), such as cGAS and LSm14A involved in recognition of viral and intracellular bacterial dsDNAs. Moreover, dsRNA-sensing protein kinase R (PKR), which has a role in antiviral immune responses in higher vertebrates, has been identified in fish. Additionally, fish possess a novel PKR-like protein kinase containing Z-DNA binding domain, known as PKZ. Here, we review the current knowledge concerning cytosolic sensors for recognition of viral and bacterial nucleic acids in teleosts.

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

Zebrafish (Danio rerio) is a laboratory model organism used in different areas of biological research including studies of immune response and host-pathogen interactions. Thanks to many biological tools available, zebrafish becomes also an important model in aquaculture research since several fish viral infection models have been developed for zebrafish. Here, we have evaluated the possible use of zebrafish to study infections with fish viruses that have not yet been tested on this model organism. In vitro studies demonstrated that chum salmon reovirus (CSV; aquareovirus A) and two alloherpesviruses cyprinid herpesvirus 1 (CyHV‐1) and cyprinid herpesvirus 3 (CyHV‐3) are able to replicate in zebrafish cell lines ZF4 and SJD.1. Moreover, CSV induced a clear cytopathic effect and up‐regulated the expression of antiviral genes vig‐1 and mxa in both cell lines. In vivo studies demonstrated that both CSV and CyHV‐3 induce up‐regulation of vig‐1 and mxa expression in kidney and spleen of adult zebrafish after infection by i.p. injection but not in larvae after infection by immersion. CyHV‐3 is eliminated quickly from fish; therefore, virus clearing process could be evaluated, and in CSV‐infected fish, a prolonged confrontation of the host with the pathogen could be studied.

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

Neutrophilic granulocytes and macrophages are crucial for the innate immune response against infections. They migrate into the focus of inflammation, where they efficiently bind, engulf and kill bacteria by proteolytic enzymes, antimicrobial peptides, reactive oxygen (ROS) and nitrogen (RNS) species. Moreover, activated neutrophils and macrophages can form extracellular traps (ETs). Fish neutrophils and macrophages are morphologically, histochemically, and functionally similar to their mammalian counterparts, but their significance for regulation of inflammatory responses and pathogen killing needs further elucidation. We compared the activity of head kidney monocytes/macrophages and neutrophilic granulocytes of common carp and established that upon lipopolysaccharide stimulation, not only neutrophils, but also carp monocytes/macrophages release extracellular DNA and are capable to form macrophage extracellular traps (METs). To clarify whether many specific LPS functions reported for piscine phagocytes might be due to impurities in the commonly used LPS preparations we studied expression of inflammatory mediators, release of DNA, ROS and RNS in cells stimulated with LPS or its highly purified form (pLPS). Also IFN-¿2 stimulation and its synergism with LPS/pLPS in stimulating expression of pro-inflammatory mediators was studied. Results substantiate that a classical stimulation of TLR4 by LPS may indeed be absent in carp as most of the classically reported LPS effects are abolished or diminished when pLPS is used. Interestingly, we also observed a potent IL-10 expression in neutrophilic granulocytes upon LPS stimulation, which, apart from their pro-inflammatory function, clearly indicates a role in restrictive control of the inflammatory reaction.

The stress response transmitted by the HPA axis is one of the best examples of neuroendocrine-immune interactions that are critical for survival. Analogous to the situation in mammals, the stress response in fish is characterized by the activation of the hypothalamo-pituitary-interrenal axis (HPI). Effects of cortisol on the fish immune system comply with findings in mammals and suggest that the differences in sensitivity to stress will influence the immune response and as a consequence of survival. Therefore, we studied the stress response and its immunity-related effects in four different carp lines (R3, R3xR8, K and R2) that display a differential pathogen susceptibility. Previous studies indicate that R3xR8 and R3 carp are susceptible to bacterial and parasite infection, while R2 and K are relatively resistant to infection. Interestingly, the most striking effect of stress on leukocyte composition and activity was observed in the pathogen-resistant K carp, even though no robust changes in gene expression of stress-involved factors were observed. In contrast, R3 carp showed no spectacular stress-induced changes in their immunological parameters with concurrent significant activation of the HPI axis. Upon stress, the R3 carp showed up-regulation of crf, pomc and gr2 gene expression in the hypothalamus. Furthermore in R3 carp, at all levels of the HPI axis, stress induced the highest up-regulation of il-1β gene expression. Although we are aware of the complexity of the interactions between stress and pathogen susceptibility and of the risk of interpretation based on correlations, it is noteworthy that the fish more susceptible to infection also exhibited the highest response to stress.

SR-A/CD204 and CD36 are major receptors responsible for oxidized lipoproteins uptake by macrophages in atherosclerotic plaques. Both receptors also share the role as receptors for different pathogens, but studies on their signaling have been hampered by the lack of selective ligands. We report that, upon specific ligation by Ab, SR-A does not induce cytokine production, but mediates inhibition of LPS-stimulated production of IL-6 and IL-12/23p40, enhancement of IL-10 release, and has no effect on TNF-α and RANTES production in murine macrophages. In contrast, anti-CD36 Ab alone stimulated production of all these cytokines, with IL-10 production being exceptionally high. Effects of anti-CD36 Ab, except of IL-10 production, were mediated by CD14 and TLR2, whereas those of SR-A ligation by heterotrimeric Gi/o proteins and by phosphatidylinositol 3-kinases. Surprisingly, we found that LPS uptake by macrophages was mediated in part by CD36 cooperating with CD14, whereas SR-A was not involved in this process. Finely, during in vitro Ag presentation to naïve CD4+ lymphocytes, pre-incubation of macrophages with anti-CD36 Ab enhanced IFN-γ production in the co-culture, but exerted the opposite effect under conditions enabling IL-10 accumulation. In contrast, anti-SR-A Ab was ineffective alone, but reversed the Th1-polarizing effect of LPS.

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

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

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

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

Due to the specific habitat conditions in which they live, earthworms are constantly exposed to pathogens. Consequently, they have evolved various immuno-defense mechanisms, including cellular (coelomocytes) and humoral responses, which may help to eliminate deleterious micro-organisms but also repair and/or protect host cells and tissues. Similar to mammalian phagocytes, coelomocytes can kill ingested pathogens with reactive oxygen species (ROS) and nitric oxide. In the present work, we studied the effects of the dermal exposure of Eisenia andrei earthworms to different immuno-stimulants: phorbol-12-myristate-13-acetate (PMA), lipopolysaccharide (LPS) or concanavalin A (ConA). After 3 days of treatment with all immuno-stimulants, decreased numbers and changed composition of the coelomocytes were observed. The immuno-stimulants also induced numerous changes in bactericidal activity, including ROS production. Furthermore, all stimulants increased cell proliferation while only LPS-treatment significantly elevated apoptosis of coelomocytes. These results demonstrate that in vivo treatment of earthworms with immuno-stimulants induces various changes in their coelomocyte response.

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.

The opioid system is involved in modulation of both innate and acquired immune responses, thus altering resistance to a variety of infectious agents. We sequenced and characterized carp opioid receptors (MOR, DOR, and KOR) and found their regulated expression in piscine leukocytes. Moreover, both in vivo and in vitro opioids affect activity of leukocytes and expression of inflammatory molecules, especially chemokines and chemokine receptors. Our data indicate an evolutionary conserved role for the opioid system in immune regulation.

Despite the discovery of many cytokine genes in fish, knowledge on their functional homology is limited. To enlighten the biological function of inflammation-related mediators, we studied their kinetics of gene expression during peritonitis in carp. Zymosan-induced intraperitoneal influx of phagocytes reached a maximum at 24 h. In peritoneal leukocytes (PTL) up-regulation of IL-1β, TNF-α, CXCa, and chemokine receptor CXCR1 preceded this peak. Delayed up-regulation of these genes in the head kidney (HK) indicates emigration of antigen-presenting cells from peritoneum into HK and/or systemic spreading of inflammation. In turn, early increase in expression of anti-inflammatory genes in HK (6 h) precede their up-regulation in the focus of inflammation. In PTL peaks of IL-10 and arginase 2 expression were recorded at 96 and 168 h, respectively. These results give evidence that carp macrophages in vivo differentiate into a continuum of different activation states with innate and alternative activation representing the extremes.

We have shown that supplementation of proinflammatory agent with a high dose of morphine not only abolishes inflammation-related pain symptoms but also inhibits influx of leukocytes to the inflamed peritoneal cavity. Present investigations focused on effects of morphine on proopiomelanocortin and prodynorphin systems during zymosan-induced peritonitis. Males of SWISS mice were ip injected with zymosan (Z, 40 mg/kg) or zymosan with morphine (ZM, 20 mg/kg). At time 0 (controls) and 4 and 24h after stimulation, peritoneal leukocytes (PTLs) were counted, PTL levels of opioid peptides (beta-endorphin and dynorphin) measured by radioimmunoassays, while mRNAs coding their respective precursors (POMC and PDYN) and receptors (MOR and KOR) determined by QRT-PCR. Influx of inflammatory PTLs, mainly PMNs, was significantly delayed by morphine co-injection. Total levels of beta-endorphin and dynorphin corresponded with PTL numbers, while levels per cell were similar in all groups except of beta-endorphin, decreased in ZM at 4h. Levels of both peptides in peritoneal fluid were increased in Z and ZM groups at 4h, while at 24h only in case of beta-endorphin in Z group. POMC was increased only in ZM group at 4h of peritonitis, while PDYN in both Z and ZM groups at the same time. MOR mRNA was increased 24h after injection in Z and ZM groups, while KOR mRNA was similar in all groups except of decrease in Z at 24h. In conclusion, endogenous opioids and their receptors are involved in zymosan-induced peritonitis and affected in various ways by morphine co-injection.

Morphine coinjection with zymosan inhibits pain and leukocyte accumulation during peritonitis in several strains of mice, and affects systems of endogenous opioids. Present investigations focus on Met-enkephalin (Met-ENK) in the inflamed peritoneal cavity and brain centers of Swiss mice. Males of Swiss mice were IP injected with zymosan or zymosan supplemented with morphine. At the selected time points the peritoneal leukocytes were counted and the Met-ENK level was measured in exudatory fluid and leukocytes, striatum, hypothalamus, and pituitary gland. The Met-ENK content in peritoneal fluid rised sharply after zymosan injection, which corresponded with its decline in exudatory leukocytes, hypothalamus, and striatum. Morphine coinjection enhanced intraperitoneal accumulation of Met-ENK and its release from exudatory leukocytes, but inhibited its early fluctuations in hypothalamus and striatum. Effects of morphine-modulated inflammation on the Met-ENK system lasted longer than 7 days.

Melatonin is responsible for the synchronization of many physiological processes, including the immune response. Here we focus on the expression of melatonin MT1 receptors in/on leukocytes, and on the effects of melatonin administration on the inflammatory processes of carp. For the first time, we showed that fish leukocytes express MT1 receptors, implicating direct responsiveness to melatonin stimulation. Moreover, both in vitro and in vivo, melatonin modulated the immune response. The most potent effects of melatonin concerned the regulation of leukocyte migration. Melatonin reduced chemotaxis of leukocytes towards CXC chemokines in vitro. In vivo, during zymosan induced peritonitis, i.p. administration of melatonin reduced the number of neutrophils. This correlated with a melatonin-induced decrease of gene expression of the CXCa chemokine. Moreover, melatonin induced a decrease of the respiratory burst in inflammatory leukocytes. Although these data do suggest a potent anti-inflammatory function for this hormone, melatonin-induced inhibition of leukocyte apoptosis clearly indicates towards a dual function. These results show that also in carp, melatonin performs a pleiotropic and extra-pineal function that is important in maintaining the delicate pro- and anti-inflammatory balance during infection. They furthermore demonstrate that neuroendocrine–immune interaction via melatonin is evolutionary conserved.

Both in mammals and fish, stress induces remarkable changes in the immune response. We focused on stress-induced changes in the activity of neutrophilic granulocytes in the R3 and K lines of common carp, which showed differential stress responses. Our study clearly demonstrates that a prolonged restraint stress differentially affects the activity of K and R3 carp neutrophils. In the K line, stress decreased the respiratory burst, while in the R3 line it reduced the release of extracellular DNA. Surprisingly, the stress-induced changes in ROS production and NET formation did not correlate with changes in gene expression of the inflammatory mediators and GR receptors. In neutrophilic granulocytes from K carp, gene expression of the stress-sensitive cortisol GR1 receptor was significantly higher than in neutrophils from R3 fish, which will make these cells more sensitive to high levels of cortisol. Moreover, upon stress, neutrophilic granulocytes of K carp up-regulated gene expression of the anti-inflammatory cytokine IL-10 while this was not observed in neutrophilic granulocytes of R3 carp. Therefore, we can hypothesize that, in contrast to R3 neutrophils, the more cortisol sensitive neutrophils from K carp respond to stress with up-regulation of IL-10 and consequently reduction of ROS production. Most probably the ROS-independent NET formation in K carp is not regulated by this anti-inflammatory cytokine. These data may indicate a predominantly ROS-independent formation of NETs by carp neutrophilic granulocytes. Moreover, they underline the important role of IL-10 in stress-induced immunoregulation.