Peptidylarginine deiminase 4 (PAD4) is a citrullinating enzyme that is gathering increasing attention due to its possible involvement in physiological processes as well as in the pathogenesis of diseases like rheumatoid arthritis or thrombosis. PAD4 is activated by calcium ions, but the details of this mechanism are elusive, because in the human body, Ca2+ concentrations are too low for full activity. Given that glycosaminoglycans (GAGs) are also implicated in the development and progression of rheumatoid arthritis, we investigated the activation of PAD4 by GAGs using heparin as a model. We employed activity assays, chromatography techniques, molecular interaction measurements (MST and SPR), FACS, and immunocytochemistry to demonstrate the activation of PAD4 by GAGs. Our data show that PAD4 binds heparin with high affinity and forms high molecular weight complexes with heparin, consistent with heparin-bound tetramer formation. Heparin activates PAD4 by increasing the enzyme’s Ca2+ affinity threefold. We also show that the effectiveness of activation with heparin depends on the length of GAG used and its negative charge. Direct measurement of heparin binding to PAD4 confirmed tight interaction with nanomolar affinity. Mutagenesis of regions likely responsible for heparin binding showed that dimerization of PAD4 is necessary for efficient activation, but the distinct binding site was not determined as interaction with heparin likely occurs over larger surface of PAD4. Furthermore, we show that other GAG family members, including heparan and chondroitin sulphates, are also able to activate PAD4. We also found that disturbed production of GAGs by CHO cells results in reduced PAD4 binding efficiency. Finally, heparin induces NETosis in hPMNs in concentration-dependent manner, as measured by the release of DNA and citrullination of histone H3. In summary, we identify the first natural coactivator of PAD4, which is present in all individuals, potentially explaining the regulation of PAD4 activity in physiological conditions, and providing new insight into the development of rheumatoid arthritis and other PAD4-related diseases. ### Competing Interest Statement The authors have declared no competing interest.

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

The physiology of ectothermic animals, including fish, is strictly regulated by season-related external factors such as temperature or photoperiod. The immune response and the production of hormones, such as estrogens, are therefore also subject to seasonal changes. This study in common carp aimed to determine how the season affects the estrogen system and the immune response, including the antibacterial response during Aeromonas salmonicida infection. We compared the immune reaction in spring and autumn in the head kidney and liver and found that carp have higher levels of blood 17β-estradiol in autumn, while in the liver of these fish there is a higher constitutive expression of genes encoding vitellogenin, estrogen receptors and Cyp19 aromatase than in spring. Fish sampled in autumn also exhibited higher expression of immune-related genes in the liver. In contrast, in the head kidney from fish sampled in the autumn, the expression of genes encoding estrogen receptors and aromatase was lower than in spring, and a similar profile of expression was also measured in the head kidney for inos , arginases and il-10 . In turn, during bacterial infection, we observed higher upregulation of the expression of inos , il-12p35 , ifnγ-2 , arginase 2 and il-10 in the liver of carp sampled in spring. In the liver of carp infected in spring a higher upregulation of the expression of the genes encoding CRPs was observed compared to fish infected during autumn. The opposite trend occurred in the head kidney, where the upregulation of the expression of the genes involved in the immune response was higher in fish infected in autumn than in those infected in spring. During the infection, also season-dependent changes occurred in the estrogen system. In conclusion, we demonstrated that season differentially affects the estrogenic and immune activity of the head kidney and liver. These results reinforce our previous findings that the endocrine and immune systems cooperate in maintaining homeostasis and fighting infection.

The circadian clock mechanism, which is evolutionarily conserved across various organisms, plays a crucial role in synchronizing physiological responses to external conditions, primarily in response to light availability. By maintaining homeostasis of biological processes and behavior, the circadian clock serves as a key regulator. This biological mechanism also coordinates diurnal oscillations of the immune response during infections. However there is limited information available regarding the influence of circadian oscillation on immune regulation, especially in lower vertebrates like teleost fish. Therefore, the present study aimed to investigate the effects of light and the timing of infection induction on the antiviral immune response in zebrafish. To explore the relationship between the timing of infection and the response activated by viral pathogens, we used a zebrafish model infected with tilapia lake virus (TiLV). Our findings demonstrated that light availability significantly affects the antiviral immune response and the functioning of the molecular clock mechanism during TiLV infection. This is evident through alterations in the expression of major core clock genes and the regulation of TiLV replication and type I IFN pathway genes in the kidney of fish maintained under LD (light-dark) conditions compared to constant darkness (DD) conditions. Moreover, infection induced during the light phase of the LD cycle, in contrast to nocturnal infection, also exhibited similar effects on the expression of genes associated with the antiviral response. This study indicates a more effective mechanism of the zebrafish antiviral response during light exposure, which inherently involves modification of the expression of key components of the molecular circadian clock. It suggests that the zebrafish antiviral response to infection is regulated by both light and the circadian clock.

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.

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.

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.

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.

Extracellular traps (ETs) are an evolutionary old mechanism of defense that functions both in higher vertebrates including mammals, lower vertebrates such as fish, in invertebrates and most probably in plants. ET structures immobilize pathogens, protect the body from their spread and possibly lead to the death of some of them. Traps formation in mammalian leukocytes is a complex process involving several molecular pathways and signaling molecules, such as reactive oxygen species (ROS), Ca2+, or protein kinases. Most probably ET formation in immunocompetent cells of non-mamalian species is subjected to similar regulations. In most cases, both in vertebrates and invertebrates, NADPH oxidase activity and consequently ROS production play an important role in this process. ET defense strategy is based on the activity of their specific components such as DNA, histones and bactericidal proteins e.g. different types of proteases. The exact composition of these structures may be slightly different in organisms belonging to different taxa, as well as depends on the type of immunocompetent cells producing the traps.

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.

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.

Neutrophils are one of the main types of effector cell engaged in the innate immune response and comprise first line of defense against pathogens. At the inflammatory site the cells eliminate pathogens and then die by apoptosis. It was commonly recognized that neutrophils utilize two ways to fight pathogens: phagocytosis and degranulation. However, in 2004 it was discovered that neutrophils can also eliminate infectious agents by formation of Neutrophil Extracellular Traps (NET) that capture and kill microorganisms. They are composed of decondensated chromatin to which specific proteins from the neutrophilic granules are attached. NETS are extracelularly released where they immobilize pathogens and secure their contact with antibacterial molecules. To date only some mechanisms of NET formation are described but it is decisively established that reactive oxygen species formed by the NADPH oxidase pathway are critical for this process. It was also reported that the release of NETs might be directly induced by pathogens or indirectly by platelets that firstly interact with microbes and subsequently with neutrophils. Altogether the published data suggest that NETs are important for pathogen elimination, however, their excessive production and/or removal might lead to abnormalities and development of severe diseases.