Full length article 17.-ethinylestradiol and 4-tert-octylphenol concurrently disrupt the immune response of common carp Magdalena Maciuszeka, Lukasz Pijanowskia, Agnieszka Pekala-Safinskab, Paulina Palichleba, Micha³ B³achuta, B.M. Lidy Verburg-van Kemenadec, Magdalena Chadzi´nskaa,* aDepartment of Evolutionary Immunology, Institute of Zoology and Biomedical Research, Faculty of Biology, Jagiellonian University, Gronostajowa 9, 30-387 Krakow, Poland bDepartment of Fish Diseases, National Veterinary Research Institute, 57 Partyzantow Ave., 24-100, Pulawy, Poland cCell Biology and Immunology Group, Wageningen University, P.O. Box 338, 6700AH, Wageningen, the Netherlands ARTICLE INFO Keywords: 17.-ethinylestradiol 4-tert-Octylphenol Inflammation Monocyte/macrophage Common carp ABSTRACT The aquatic environment is massively polluted with endocrine-disrupting compounds (EDCs) including synthetic estrogens (e.g. 17.-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-.2 and IFN-.-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-.2 was found in the focus of inflammation. Moreover, during A. salmonicida- induced infection in EE2-treated carp, but not in fish fed with 4t-OP-treated food, we found an enhanced inflammatory reaction manifested by high number of inflammatory peritoneal leukocytes, including phagocytes and higher expression of pro-inflammatory mediators (inos, il-1ß, cxcl8_l2). Furthermore, in the liver, EE2 down- regulated the expression of acute phase proteins: CRPs and C3. Importantly, both in vitro and in vivo, EDCs altered the expression of estrogen receptors: nuclear (er. and erß) and membrane (gpr30). EDCs also induced up- regulation of the cyp19b gene. Our findings reveal that contamination of the aquatic milieu with estrogenic EDCs, may considerably violate the subtle and particular allostatic interactions between the immune response and endogenous estrogens and this may have negative consequences for fish health. 1.Introduction The aquatic milieu is particularly exposed to contamination as it forms a major sink for anthropogenic substances; industrial bulk chemicals, predominantly causing non-specific modes of toxic action, but also (emerging) micropollutants with specific biological modes of action [1]. They include endocrine-disrupting compounds (EDCs) such as synthetic estrogens (e.g. 17.-ethinylestradiol, EE2), alkylphenols (e. g. 4-tert-octylphenol, 4t-OP), plasticizers (e.g. bisphenol A, BPA) and a number of pesticides that may be formed e.g. during the production of fertilizers or paint [1]. EE2 is one of the more potent pro-estrogenic EDCs used primarily in contraceptives and in hormone replacement therapy [2]. It is continuously released into surface waters via sewage treatment plants [3]. In turn, 4t-OP is one of the major biodegradation products of nonionic surfactants: alkylphenol polyethoxylates, which are released to the environment during production of detergents, paints, plastics, herbicides, and pesticides, and have become a major component in wastewater systems [4]. Octylphenol shows marked similarities to natural estrogens and has higher estrogenic potency compared to other alkylphenols of the same class [5,6]. The xenoestrogens, although chemically different from the estrogenic hormones produced internally by the endocrine system, have *Corresponding author. E-mail address: magdalena.chadzinska@uj.edu.pl (M. Chadzi´nska). Contents lists available at ScienceDirect Fish and Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi https://doi.org/10.1016/j.fsi.2020.10.005 Received 23 June 2020; Received in revised form 30 September 2020; Accepted 3 October 2020 Imprint logo Journal logo estrogenic effects in the organism [7–9]. They can bind to nuclear (ER) and membrane (GPR30) estrogen receptors, as well as to peroxisome proliferator-activated receptors (PPARs) and aryl hydrocarbon receptors (AhR). They may thus act as agonists or antagonists. Moreover, estrogenic EDCs interact with other components of the endocrine pathway, e. g. ER corepressors or coactivators and modulate the activity of the enzymes involved in steroid conversion e.g. aromatase CYP19 that is responsible for the conversion of testosterone to 17ß-estradiol (E2). This leads to stimulation or inhibition of endogenous hormone biosynthesis. Furthermore, EDCs bind to circulating hormone-binding proteins and disrupt the local endogenous hormonal balance e.g. of estradiol or cortisol [8]. Regulation of sexual differentiation and reproduction is the key physiological role of estrogens. Therefore, estrogenic EDCs are mainly viewed as reproductive disruptors and consequently studies predominantly focused on the disbalance in reproductive parameters they induce, e.g. raised levels of egg yolk protein precursor - vitellogenin in male fish [10]. However, it has been often overlooked that EDCs have pleiotropic activities and interfere with other physiological processes regulated by endogenous estrogens, such as growth, osmoregulation and immune reactions [11–16]. We and others proved that leukocytes express ER and GPR30 estrogen receptors [15,17–19] and that endogenous estrogens e.g. E2 exert immunomodulatory functions [17,20–22]. In mammals, low E2 concentrations, e.g., in post-menopausal women, promote Th1 type immune responses to favor cell-mediated immunity and production of pro-inflammatory cytokines (e.g. TNF-., IFN-.). In contrast, high E2 concentrations, occurring for example during pregnancy, promote a Th2-immune response, production of cytokines such as IL-4, IL-10 and IL-13 and antibody production [21,23]. In turn in fish, E2 affects phagocytosis and production of reactive oxygen species (ROS) in phagocytes [19,24,25] and alters adaptive immune responses e.g. antibody levels and mitogen-induced proliferation of lymphocytes [26, 27]. As a consequence, E2-induced modulation of fish susceptibility to infection was observed [28–30]. In this manner the neuroendocrine and immune systems coordinate their actions to ensure allostasis during altering environmental conditions like season or reproductive cycle. There is good evidence that the hypothalamus-pituitary-gonad axis-immune crosstalk is part of the proximate mechanisms underlying the reproductive-immune trade-offs of vertebrates (for review Segner and others [31]). In the present work we studied the effects of two estrogenic EDCs: EE2 and 4t-OP on the anti-bacterial immune response of common carp. Most of the EDCs that are released by industry, agriculture and medicine cannot be eliminated by conventional techniques of water treatment (e. g. Ref. [32]) and they have a tendency to accumulate in the sediment [33]. Carp are considered extra vulnerable as they mainly feed on the bottom fauna and therefore are exposed to higher doses of EDCs than other fish. We were particularly interested in the effects of EDCs on the activity of macrophages because both in mammals and fish, macrophages are crucial immunocompetent cells for the innate immune response. They migrate to the site of inflammation to eliminate pathogens through phagocytosis and produce ROS, NO, enzymes (e.g. lysozyme) and antimicrobial peptides that are all toxic for pathogens [34,35]. Furthermore, macrophages present foreign antigen to T-cells to initiate a cellular adaptive immune response. In this response, activated macrophages release cytokines such as IL-12 and IL-1ß that stimulate Th1 cells to produce IFN-. which in turn stimulates macrophages to produce higher amount of NO. IFN-. also stimulates leukocytes to produce CXCL9-11 chemokines e.g. CXCb in common carp [36,37]. We studied EDC effects on the absolute and relative numbers of inflammatory leukocytes and on the expression of mediators involved in the initiation of an inflammatory response and pathogen eradication. The latter include pro-inflammatory cytokines: IL-1ß, IL-12, IFN-.2, chemokines that direct leukocyte migration: CXCL8_L2 and CXCb2, and the enzyme: inducible nitric oxide synthase (iNOS) that catalyzes the synthesis of nitric oxide (NO). Moreover, we measured the expression/ activity of the anti-inflammatory IL-10 and molecules involved in the process of wound healing and tissue regeneration such as arginase 1 and 2 and matrix metalloproteinase 9 – MMP9. In the liver the expression of the acute phase proteins: C-reactive protein (CRP) and the C3 complement protein were studied, both involved in pathogen eradication and in host tissue protection during inflammation. Moreover, to determine the mechanisms responsible for the effects of EDCs, we also studied the expression of their putative receptors and of aromatase Cyp19. 2.Materials and methods 2.1.Animals Sexually immature, young (9–12 months) common carp (Cyprinus carpio L.) from line R3xR8 were obtained from the Institute of Ichthyobiology and Aquaculture, Polish Academy of Science, Golysz, Poland. Fish used for treatment groups within one experiment were always from the same breed and to limit potential differences between experiments we always use fish of the same age category, when they have an appropriate size and have a well-developed mature immune system. After transport, fish were adapted for 4 weeks to new living conditions at the Institute of Zoology and Biomedical Research in Krakow, Poland. Fish were kept in tanks with recirculating tap water (volume 375 L, flow rate 4 L/min, density 45 fish/tank and 60 g/L) at 21 .C and fed with dry food (Aller Master, Aller Aqua, Poland) - 1% of the body weight/day. Animals were handled in strict accordance with good animal practice as defined by the national and local animal welfare bodies, and procedures were approved by the local ethical committee (2nd Local Institutional Animal Care and Use Committee (IACUC) in Krakow, Poland, license number 291/2017). After acclimation, fish were randomly divided into 3 different tanks/ treatments groups (7 fish per tank/treatment group): control – fish fed with control food (fish weight: 83.39 ±4.63 g), EE2 – fish fed with food treated with 17.-ethinylestradiol (EE2, 50 mg/kg, Sigma-Aldrich, St. Louis, MO, USA) (fish weight: 81.86 ±7.19 g) and 4t-OP – fish fed with food treated with 4-tert-octylphenol (4t-OP, 2.5 µg/day/fish 4t-OP/kg food, Sigma-Aldrich, St. Louis, MO, USA) (fish weight: 80.61 ±4.70 g). The experiment was performed two times, so finally 14 fish were analyzed for each treatment group. The feed preparation and choice of EE2 and 4t-OP concentrations were described before [38,39] and were designed to widely cover the range of actual pollution levels that may be reached in the environment. They induce a pro-estrogenic effect, as shown by up-regulation of vtg gene expression in the liver and 17ß-estradiol in blood serum (Fig. S1) and at the same time do not cause mortality or lesions in not-infected fish. Commercially available dry food (Aller Master, Aller Aqua, Poland) was spiked either with EE2 or 4t-OP diluted in ethanol (99.8%, POCH, Gliwice, Poland) and processed by the alcohol evaporation method [40] with some modification: food was dried for 2 h at room temperature. Control food was spiked with the same volume of ethanol alone. Fish were fed at a daily maintenance rate of 1% of their estimated body weight for 14 days. At the day of sacrifice (at 24 and 96 hpi) fish weight did not differ significantly between control and treatment groups. At 24 hpi fish from the control group weighed 90.86 ±5.86 g, from the EE2 group – 97.79 ±8.09 g and from the 4t-OP group – 85.36 ±7.17 g. At 96 hpi fish from the control group weighed 89.93 ±5.49 g, from the EE2 group – 85.42 ±9.60 g and from the 4t-OP group – 85.62 ±5.95 g. 2.2.Infection A.salmonicida subsp. salmonicida from Polish origin was obtained from the Department of Fish Diseases, National Veterinary Research Institute, Pulawy. Bacteria were grown in lysogeny broth (LB) medium M. Maciuszek et al. for 18 h at 25 .C, centrifuged at 1600 g for 10 min and the bacterial pellet was reconstituted in sterile PBS (280 mOsM). The optical density was measured at 625 nm and data were aligned with a previously derived McFarland scale to determine the bacterial concentration. On day 14 of feeding, fish from control, EE2 and 4t-OP groups (n =14) were injected intraperitoneally (i.p.) with a non-lethal dose of A. salmonicida (4 x 108 bacteria in 250 µL PBS per fish) as described previously [41]. At 24 and 96 h post-injection (hpi) fish were anaesthetized with tricaine methane sulphonate (TMS; Sigma-Aldrich, St. Louis, MO, USA; 0.2 g/L) buffered with NaHCO3 (POCH, Gliwice, Poland; 0.4 g/L). 2.3.Serum hormone level Fish were bled and blood was collected in covered test tubes and allowed to clot at 4 .C for 12 h. Blood serum was collected, and hormone levels were determined with commercial kits according to the manufacturer’s protocol. Estradiol level was measured with a DRG kit (Marburg, Germany; range 10.6–2000 pg/mL, sensitivity 10.60 pg/mL) while cortisol was measured with the Neogen Kit (Lexington, USA; range 0.04–10.0 ng/mL; sensitivity 0.04 ng/mL). 2.4.Organ and cell isolation After the bleeding, peritoneal leukocytes (PTL) were taken by flushing the peritoneal cavity with 2 mL of sterile PBS (280 mOsM) with heparin (Sigma-Aldrich, St. Louis, MO) as described previously [42]. To determine PTL composition, phagocytes (mononuclear and polymorphonuclear leukocytes) and lymphocytes were counted in a hemocytometer after Türk staining as described previously [42]. Organs (head kidney and liver) were carefully removed and immediately transferred to fix RNA (Eurex, Gdansk, Poland) and kept at 20 .C for further analysis. To determine the cell composition of the head kidney leukocytes, organs were passed through a 100 µm nylon mesh with carp RPMI, (cRPMI 1640, Invitrogen, Carlsbad, CA, adjusted to carp osmolarity of 270 mOsm/kg with distilled water) containing heparin (Sigma-Aldrich, St. Louis, MO) and washed once and resuspended in cRPMI. Cell composition was analyzed with a FACSCalibur flow cytometer (BD Biosciences) as described previously by Kepka et al. [43]. 2.5.In vitro cell isolation and stimulation The monocyte/macrophage enriched suspensions from the head kidney were obtained as described previously by Maciuszek et al. [44]. After isolation cells were resuspended in carp cRPMI++(cRPMI supplemented with 0.5% (v/v) pooled carp serum with antibiotics (1% L-glutamine (Sigma-Aldrich, St Louis, MO, USA), 1% (v/v) penicillin G (Sigma Aldrich, St Louis, MO, USA) and 1% (v/v) streptomycin sulphate (Sigma-Aldrich, St Louis, MO, USA)) to a density of 10 million cells per ml. To determine gene expression, monocytes/macrophages were seeded in 24-well cell culture plates (Nest Biotech Co, Wuxi, China) at 27 .C, 5% CO2 and were treated either with EE2 (0.1, 1 and 10 µM, Sigma-Aldrich, St. Louis, MO, USA), 4t-OP (0.1, 1 and 10 µM), LPS (LPS, Escherichia coli serotype O55: B5, Sigma-Aldrich, St. Louis, MO, USA; L2880, 30 µg/mL) or their combination (EE2+LPS or 4t-OP+LPS). EE2, 4t-OP and LPS concentrations were chosen based on previous studies [17,45,46]. Control cells (CTR) were treated with the same volume of culture medium. After 6 h of stimulation, cells were resuspended in 350 µL RL buffer (Eurex, Gdansk, Poland) with 1% (v/v) ß2-mercaptethanol (Sigma-Aldrich, St. Louis, MO, USA) and kept at 80 .C for further analyses. To determine cell activity (arginase activity and NO production), monocytes/macrophages were seeded in 96-well cell culture plates (Nest Biotech Co, Wuxi, China) at 27 .C, 5% CO2 and stimulated for 24 h with EE2, 4t-OP, LPS or their combinations (EE2+LPS or 4t-OP+LPS). The in vitro experiments were performed 3 times independently and each time monocyte/macrophage primary cultures were derived from 1 or 2 fish. 2.6.Arginase activity and nitric oxide production Following 24 h stimulation, arginase activity was measured in cell lysates as described previously by Corraliza and co-workers [47], with small modifications described by Maciuszek et al. [44]. Nitrite/nitrate production, an indicator of NO synthesis, was measured in cell culture supernatants as described previously [48]. 2.7.Gene expression 2.7.1.RNA isolation and cDNA synthesis RNA was isolated from cells and tissues with GeneMATRIX Universal RNA Purification Kit (Eurex, Gdansk, Poland) according to the manufacturer’s protocol. Final elution was carried out in 30 µL of nuclease- free water. Before proceeding with further analyses, RNA was quantified and its integrity checked (Tecan Spark NanoQuant PlateTM.). The cDNA synthesis was performed with High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Waltham, Massachusetts, USA) according to the manufacturer’s protocol. 2.7.2.Real-time quantitative PCR Carp-specific primers (5'–3') for immune-related (inos, il-1ß, il-12p35 ifn-.2, cxcl8_l2, cxcb2, c3, crp1, crp2, il-10, arginase 1, arginase 2, mmp9) and endocrine-related (er., erß, eer., eerß, gpr30, err., cyp19a, cyp19b, vitellogenin, ppar.) RNA detection were used. The 40S ribosomal protein s11 (40s11) gene served as an internal standard. Accession numbers and primer sequences are listed in Table S1. RT-qPCR was performed as described previously [44] and changes in the gene expression upon fish or cell treatment were rendered as a ratio of target gene vs. reference gene (40S ribosomal protein s11 gene) relative to the expression in control samples according to the method described by Pfaffl, 2001 [49]. 2.8.Statistical analysis Data were expressed as mean and standard error (SE). Significances of differences were compared by two-way analysis of variance (ANOVA), followed by post hoc Tukey’s test. The differences were considered statistically significant at p <0.05. 3.Results 3.1.EDC-induced changes in monocyte/macrophage activity in vitro In vitro EE2 did not affect the gene expression of pro-inflammatory mediators in unstimulated cells while in LPS-stimulated cells, 0.1 µM of EE2 up-regulated the gene expression of il-1ß (Fig. 1B) and il-12p35 (Fig. 1D). In contrast, a 10 µM concentration of EE2 down-regulated the expression of il-12p35 and cxcb2 (Fig. 1D and F). Similarly, a 10 µM concentration of 4t-OP down-regulated the expression of il-12p35, ifn-.2 and cxcb2 in LPS-stimulated monocytes/macrophages (Fig. 2D, E and F). Moreover, 4t-OP (10 µM), but not EE2, decreased the levels of NO released from LPS-stimulated cells (Fig. 3B). Both EDCs did affect the expression and activity of the anti-inflammatory mediators (Figs. 1 and 2 G-J and Fig. 3C and D). In vitro, EE2 down-regulated the erß gene expression in LPS- stimulated monocytes/macrophages. In unstimulated monocytes/macrophages 4t-OP up-regulated the erß gene expression. Both EDCs did not affect gene expression of er., err., errß, ahr, ppar. and cyp19a and b (Table S2), while gene expression of the estrogen-related receptor gamma (eer.) was absent in monocytes/macrophages of carp. ppar. gene expression in LPS-stimulated monocytes/macrophages M. Maciuszek et al. was down-regulated, while its expression remained unchanged upon in vitro treatment with EDCs (Fig. S2). 3.2.EE2 and 4t-OP show estrogenic activity At 24 hpi, both EE2 and 4t-OP induced up-regulation of gene expression of vitellogenin in the liver. The EE2-induced increase was higher than that induced with 4t-OP. Up-regulation of vitellogenin expression was also observed at 96 hpi in EE2 treated fish (Fig. S1A). Moreover, EE2 increased the serum level of 17ß-estradiol at 24 hpi while 4t-OP-induced up-regulation of the E2 level at 96 hpi (Fig. S1B). In fish treated with EE2, at 24 hpi also increased levels of serum cortisol were found (Fig. S1C). 3.3.EDC-induced in vivo changes in immune response in the peritoneum, head kidney and liver No mortality was observed during the feeding and infection periods. Fig. 1.In vitro effects of 17 . -ethinylestradiol on gene expression of pro-inflammatory mediators and CXC chemokines (A–F) and on anti-inflammatory mediators (G–J) in monocytes/macrophages. Cells were in vitro treated for 6 h with lipopolysaccharide (LPS, 30 µg/mL) and/or 17 . -ethinylestradiol (0.1 µM, 1 µM, 10 µM). Changes in gene expression are shown as x-fold increase compared to un-stimulated cells that are treated with culture medium (CTR, 0 µM). They were standardized for the housekeeping gene 40S ribosomal protein s11. Averages and S.E (n =5–6). Mean values not sharing letters are statistically different. M. Maciuszek et al. Image of Fig. 1 Moreover, upon A. salmonicida injection, control and 4t-OP-treated fish did not show any symptoms of infection (Figs. S3A and D) while in EE2- treated fish, Aeromonas salmonicida caused skin lesions (Figs. S3B and C). Furthermore, in EE2-treated fish at 24 hpi, higher numbers of peritoneal leukocytes, including phagocytes, were observed compared to control fish at the same time point of infection. In contrast, in 4t-OP- treated fish, numbers of peritoneal leukocytes/phagocytes were lower compared to those in control infected animals (Fig. 4). While in control animals numbers of peritoneal leukocytes were decreased at 96 hpi compared to 24 hpi, the percentage of granulocytes and lymphocytes in the head kidney increased (Fig. 4D). However, the relative leukocyte composition in the head kidney was not affected by EDC-treatment at both time points. At 24 hpi, in the EE2-treated fish a higher expression of pro- inflammatory (il-1ß, cxcl8_l2) and anti-inflammatory (arginase 2, il-10 and mmp-9) mediator genes was observed in the peritoneal leukocytes, while at this time point EE2 decreased the expression of ifn-.2 (Fig. 5). Fig. 2.In vitro effects of 4-tert-octylphenol on the gene expression of pro-inflammatory mediators and CXC chemokines (A–F) and on anti-inflammatory mediators (G–J) in monocytes/macrophages. Cells were in vitro treated for 6 h with lipopolysaccharide (LPS, 30 µg/mL) and/or 4-tert-octylphenol (0.1 µM, 1 µM, 10 µM). Changes in gene expression are shown as x-fold increase compared to un-stimulated cells, treated with culture medium (CTR, 0 µM) and they were standardized for the housekeeping gene 40S ribosomal protein s11. Averages and S.E (n =5–6). Mean values not sharing letters are statistically different. M. Maciuszek et al. Image of Fig. 2 Moreover, gene expression of inos, il-1ß, cxcl8_l2 and il-10 was higher at 96 hpi in EE2-treated animals than in control infected fish (Fig. 5). In contrast, at 96 hpi EE2 decreased the expression of il-12p35 (Fig. 5D). Similarly, at this stage of infection, gene expression of il-12p35, ifn-.2 and cxcb2 was lower in 4t-OP-treated fish than in control animals (Fig. 5D, E and F). In the head kidney, EE2-treatment increased the level of arginase 2 at 24 hpi and of il-12p35 and arginase 1 mRNA at 96 hpi, while 4t-OP did not affect gene expression of inflammatory mediators in this organ, with exception of the up-regulation of mmp9 gene expression at 24 hpi (Table S3). At 24 hpi, in the liver of EE2-treated fish, an increased expression of the inos, il-1ß and arginase 2 genes was found compared to control fish (Fig. 6A, B, E). 4t-OP did not affect the expression of these genes. In contrast both at 24 and 96 hpi, EE2 down-regulated expression of c3 and crp1 in the liver (Fig. 6F and G). Moreover, in EE2-treated animals down- regulation of crp2 gene expression was found at 96 hpi (Fig. 6H). 3.5.EDC-induced in vivo changes in the expression of estrogen receptors and aromatase CYP19 At 24 hpi in EE2-treated fish higher gene expression of cyp19b in peritoneal leukocytes (Fig. 7E) and er. in the liver (Fig. 7K) was found. Moreover, at this time point 4t-OP up-regulated gene expression of erß in peritoneal leukocytes (Fig. 7B). At 96 hpi EE2 induced up-regulation of er. (Fig. 7A), gpr30 (Fig. 7C) and cyp19b (Fig. 7E) gene expression in peritoneal leukocytes. At this time point of infection, EE2 induced up-regulation of erß (Fig. 7G), gpr30 (Fig. 7H), and both cyp19 genes in the head kidney (Fig. 7I and J). Up- regulation of cyp19b gene expression was also found at 96 hpi in peritoneal leukocytes and liver of 4t-OP-treated fish (Fig. 7E and O). Both EE2 and 4t-OP did not affect the expression of ppar. in the liver (data not shown). 4.Discussion The endocrine disruptors we studied are xenoestrogens that can be found in European sewage and waters. They induce adverse effects on the endocrine system of several organisms, including fish. Since EDCs can affect physiological systems beyond the endocrine system, we assessed in vitro and in vivo effects of EE2 and 4t-OP chemical species on the carp immune response. We found that in monocytes/macrophages, a low concentration of EE2 up-regulated the LPS-induced expression of the pro-inflammatory il-1ß and il-12p35 genes. In contrast, high concentration of both EE2 and 4t-OP down-regulated IFN-.2 expression and the IFN-.-dependent immune response in LPS-stimulated cells. Also, upon in vivo EE2 and 4t-OP treatment of fish that suffered from a bacterial infection, a decreased gene expression of il-12p35 and ifn-.2 was found in the peritoneal leukocytes. Moreover, gene expression of IFN- .-inducible CXCb2 chemokine was lower in peritoneal leukocytes of 4t- OP-treated fish than in infected control animals. These data suggest that estrogenic EDCs modulate the Th1-driven cellular immune response of fish. In the literature, reports of the effects of EDCs on T cells are often contradictory and depend on the animal species, EDC concentration, and time of exposure, or type of EDC [50]. For example, in line with our observation, another estrogenic EDC, BPA, inhibited the biochemical pathways that are related to Th1 immune responses in phytohemagglutinin-stimulated human peripheral blood mononuclear cells [51]. In turn, in mice, lower doses of BPA increased the expression of inf., tnf., and il-6, while higher doses decreased their expression [52]. Moreover, EDCs inhibited the LPS-induced production of NO by macrophages, both in in vivo animal studies and in in vitro studies of cell cultures. Yoshitake et al. [53] found that BPA, nonylphenol (NP) and 4t-OP suppressed the LPS-induced NO production in the murine Fig. 3.In vitro effects of 17 . -ethinylestradiol and 4-tert-octylphenol on nitric oxide production (A–B) and on arginase activity (C–D) in head kidney monocytes/ macrophages. Cells were in vitro treated for 24 h either with lipopolysaccharide (LPS, 30 µg/mL), and/or 17 . -ethinylestradiol (0.1 µM, 1 µM, 10 µM), 4-tert- octylphenol (0.1 µM, 1 µM, 10 µM) or with culture medium (CTR, 0 µM). Averages and S.E (n =7–8). Mean values not sharing letters are statistically different. M. Maciuszek et al. Image of Fig. 3 macrophage cell line RAW264.7 and these effects were blocked by the ER inhibitor. Moreover, NO production was also reduced in RAW264.7 macrophages after treatment with EE2 [54]. In fish, corresponding to our results, Cabas and colleagues [55] found that in vitro, low concentrations of EE2 increased the mRNA levels of pro-inflammatory il-1ß, il-6, tnf. in macrophages from the head kidney of gilthead seabream, while higher doses of EE2 decreased the production of reactive oxygen species and the expression of pro-inflammatory mediators (il-1ß, il-6, cox2) in leukocytes stimulated with bacterial DNA. At the same time, EE2 increased the expression of il-8, mmp9 and mmp13 [55]. In contrast, EE2 failed to affect NO release while it reduced DNA-stimulated up-regulation of the above-mentioned genes [56]. Authors proposed that estrogens, by activating endothelial cells, modulate fish leukocyte trafficking during the inflammatory process. Also, in our previous experiments, E2 down-regulated the LPS-induced expression of il-12p35 and cxcb2 in vitro [20]. These results can be, at least partially, explained by the fact that both in mammalian and fish macrophages, E2 and EDC induced a decrease in the expression of the nuclear transcription factor nf-.b, which is a crucial regulator of the production of numerous inflammatory mediators including IL-12 [57–60]. Surprisingly, 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 a high number of inflammatory peritoneal leukocytes, including phagocytes, and a higher expression of pro-inflammatory mediators (inos, il-1ß, cxcl8_l2). However, in peritoneal leukocytes of these animals we also found higher expression of genes encoding anti-inflammatory il-10 and two genes encoding enzymes involved in tissue remodeling: arginase 2 and mmp-9. Moreover, EE2 intensified the appearance of severe skin lesions. In EE2-treated animals, liver enlargement and tissue hyperemia were also observed (Fig. S4). Therefore, we can conclude that EE2 dysregulated the anti- bacterial immune response of carp. In line with these results, in not-infected medaka and common carp, EE2 exposure caused histopathological alterations and severe hemorrhages in kidney, liver and spleen [61,62]. Moreover, exposure to EE2-rich diet promoted a long-term peritonitis in gilthead seabream, both in control and in animals treated with hemocyanin [63]. In this case EE2 was able to stimulate leukocyte recruitment to the focus of inflammation. It also elevated the production of reactive oxygen species and the expression of il-1ß in the peritoneal leukocytes from unvaccinated fish. In the present study, up-regulation of inos, il-1ß, and arginase 2 was also found in the liver of infected fish that were treated with EE2. But next to the EE2-induced up-regulation of inflammatory mediators, EE2 down-regulated the expression of the acute phase proteins CRPs and C3 in the liver. Earlier, Wenger and colleagues [29] found that in rainbow trout, E2 in vivo affected the gene expression of complement factors during a Yersinia ruckeri infection. In these E2-treated animals, the infection-induced up-regulation of the mRNA levels of C3-1, C3-3. The factor B and factor H genes were reduced. Furthermore, in the liver of EE2-treated male medakas that were infected with Edwardsiella tarda, down-regulation of genes involved in pathogen recognition (tlr3, tl5s, myd88), lysozyme (lyz) and complement c3 factor was found, while in infected females treated with EE2, only tlr3 and c3 were downregulated [64]. In the liver of male medakas, EE2 also Fig. 4.In vivo effects of 17 .-ethinylestradiol or 4-tert-octylphenol on the number and composition of peritoneal (A–C) and head kidney (D) leukocytes. Fish were fed for 14 days with control food (CTR) or food treated with 17 .-ethinylestradiol (EE2, 50 mg/kg) or 4-tert-octylphenol (4t-OP, 2.5 µg/day/fish 4t-OP/kg food). On day 14 of feeding, fish were injected i.p. with A. salmonicida (4 x 108 bacteria in 250 µL PBS per fish). At 24 and 96 h post-infection (hpi) peritoneal and head kidney leukocytes were collected, counted and their composition was analyzed. Averages and S.E (n =14). Mean values not sharing letters are statistically different. M. Maciuszek et al. Image of Fig. 4 decreased the gene expression of two transcription factors that are important for the immune defense: nfkb and stat3, and this inhibition could be reversed by an ER. antagonist [65]. Moreover, microarray analysis of gene expression in the liver of common carp that were treated with E2, NP, BPA or EE2, demonstrated that EDCs regulate the expression of genes responsible for development, reproduction, metabolism and for the immune system, including a concentration-dependent inhibition of multiple genes encoding subunits of the complement pathway [66]. Similar results were obtained for largemouth bass, where EE2 affected the expression of hepatic complement components [67]. Also, transcriptomic analysis of rainbow trout liver revealed that EE2 treatment induced down-regulation of genes involved in the inflammatory response [68]. In this context, it has to be mentioned that in mammals, many immune genes, including complement factor C3, are directly regulated by estrogens as they have a functional estrogen-responsive element (ERE) in the promoter region [69]. To date, corresponding data for fish are limited. To clarify the mechanisms that mediate the effects of EDCs on the fish immune system, we examined the expression of genes encoding classical estrogen receptors and other putative receptors binding EE2 Fig. 5.In vivo effects of 17 .-ethinylestradiol or 4-tert-octylphenol on gene expression of pro-inflammatory mediators and CXC chemokines (A–F) and on anti- inflammatory mediators (G–J) in peritoneal leukocytes. Fish were fed for 14 days with control food (CTR) or food treated with 17 .-ethinylestradiol (EE2, 50 mg/kg) or 4-tert-octylphenol (4t-OP, 2.5 µg/day/fish 4t-OP/kg food). On day 14 of feeding, fish were injected i.p. with A. salmonicida (4 x 108 bacteria in 250 µL PBS per fish). At 24 and 96 h post-infection (hpi) the peritoneal leukocytes were collected, and gene expression was measured. Changes in gene expression are shown as x- fold increase compared to the control group (CTR) and standardized for the housekeeping gene 40S ribosomal protein s11. Averages and S.E (n =14). Mean values not sharing letters are statistically different. M. Maciuszek et al. Image of Fig. 5 and 4t-OP. We neither observed EDC-induced changes in the expression of genes encoding estrogen-related receptor (ERR), AhR, nor peroxisome proliferator-activated receptor (PPAR.). In the last case, significant downregulation of ppar. was found in monocytes/macrophages upon LPS stimulation. Similar changes in ppar. expression upon LPS treatment, were previously found for example in the RAW264.7 cell line [70, 71] and in murine peritoneal macrophages [72]. However, a few studies also indicated that expression of these receptors can be changed upon EDCs. For example, PPAR. mRNA levels increased at 1 µM and decreased at 50 µM of EE2 in brown trout hepatocytes [73]. Moreover, EE2-induced up-regulation of expression of the esr1 gene in hepatocytes of several fish species [11,74–76]. In contrast, no change in the expression of esr1 was observed in hepatocytes of EE2-treated largemouth bass [67]. Importantly, upon EDC treatment we observed a slight but significant change in the expression of classical nuclear estrogen receptors suggesting that this kind of estrogen receptors is involved in EDC- induced immunoregulation. In vitro, EE2 down-regulated the expression of erß in LPS-stimulated monocytes/macrophages, while 4t-OP up- regulated the expression of this gene in unstimulated cells. More complex changes in the gene expression of estrogen receptors were observed upon in vivo EDC treatment. In peritoneal leukocytes, EE2 up-regulated the expression of er. and gpr30 at 96 hpi, whereas 4t-OP up-regulated the expression of erß at 24 hpi. At 96 hpi, in the head kidney of EE2- treated fish we found a higher expression of erß and gpr30. In the liver, EE2 induced higher expression of the er. gene at 24 hpi. Previously, Cabas and others [55] found that EE2 induced the up-regulation of era expression in the head kidney of gilthead seabream, while Liarte and colleagues [77] observed that in this species EE2 increased the expression of the era gene in endothelial cells that were stimulated with bacterial DNA. Moreover, EE2-induced increase of gene expression of era and gpr30 was found in peritoneal exudate leucocytes of gilthead seabream [63]. In turn, studies in rodent macrophages suggested that the ER.-dependent inhibition of NF-.B is the main pathway involved in EDC-induced changes in cytokine expression [78]. As some studies highlight that EDC actions can be linked to changes in steroid synthesis and metabolism (for review Casals-Casas and Desvergne [9], Cheshenko and others [79], we decided to measure the effects of EE2 and 4t-OP on the expression of genes encoding CYP19 aromatase. This enzyme is critical for the conversion of C19 steroids to estrogens. In fish, two aromatase genes were discovered: cyp19a, mainly expressed in ovaries, and cyp19b, displaying the highest expression in the brain [80]. In our previous studies we found that both cyp19 genes are also constitutively expressed in the lymphoid organs and different populations of leukocytes of common carp [81]. In the present experiments, Fig. 6.In vivo effects of 17 .-ethinylestradiol or 4- tert-octylphenol on gene expression of pro- inflammatory mediators (A–C), anti-inflammatory mediators (D–E) and acute phase proteins (F–H) in the liver. Fish were fed for 14 days with control food (CTR) or food treated with 17 .-ethinylestradiol (EE2, 50 mg/kg) or 4-tert-octylphenol (4t-OP, 2.5 µg/day/ fish 4t-OP/kg food). On day 14 of feeding, fish were injected i.p. with A. salmonicida (4 x 108 bacteria in 250 µL PBS per fish). At 24 and 96 h post-infection (hpi) the livers were collected and gene expression was measured. Changes in gene expression are shown as x-fold increase compared to the control group (CTR) and standardized for the housekeeping gene 40S ribosomal protein s11. Averages and S.E (n =14). Mean values not sharing letters are statistically different. M. Maciuszek et al. Image of Fig. 6 Image of Fig. 7 Fig. 7.In vivo effects of 17 .-ethinylestradiol or 4-tert-octylphenol on gene expression of estrogen receptors and aromatase CYP19 in the in peritoneal leukocytes (A–E), in the head kidney (F–J) and in the liver (K–O). Fish were fed for 14 days with control food (CTR) or food treated with 17 .-ethinylestradiol (EE2, 50 mg/kg) or 4-tert -ctylphenol (4t-OP, 2.5 µg/day/fish 4t-OP/kg food). On day 14 of feeding, fish were injected i.p. with A. salmonicida (4 x 108 bacteria in 250 µL PBS per fish). At 24 and 96 h post-infection (hpi) the peritoneal leukocytes, head kidneys and livers were collected, and gene expression was measured. Changes in gene expression are shown as x-fold increase compared to the control group (CTR) and standardized for the housekeeping gene 40S ribosomal protein s11. Averages and S.E (n =14). Mean values not sharing letters are statistically different. M. Maciuszek et al. in all tissues studied, EDCs induced up-regulation of expression of the cyp19b, but not the cyp19a gene. These data are consistent with the fact that structural analysis of the cyp19a and cyp19b gene promoters showed the presence of EREs in the cyp19b promoter only [80,82]. Our findings reveal that, where the endogenous estrogens act as physiological mediators that cooperate in the complex regulation of the immune response, increasing contamination of the aquatic milieu with estrogenic EDCs may considerably violate these subtle and precise allostatic interactions. Especially the dysregulation of the important Th1 pathway may induce consequent negative effects for fish health. CRediT authorship contribution statement Magdalena Maciuszek: c-designed and performed the experiments, collected, analyzed and discussed data and wrote the manuscript, Formal analysis, Writing - original draft. Lukasz Pijanowski: performed some experiments. Agnieszka Pekala-Safinska: provided A. salmonicida. Paulina Palichleb: performed some experiments. Micha³ B³achut: and. B.M. Lidy Verburg-van Kemenade: discussed the data and co-wrote the manuscript, Writing - original draft. Magdalena Chadzi´nska: co-designed experiments supervised the study and co-wrote the manuscript, Writing - original draft. Acknowledgements We like to thank Dr. Elena Wernicke-von Siebenthal for valuable advices for food preparation. Appendix A.Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.fsi.2020.10.005. Funding This work was supported by the Polish National Science Center (grant no. 2015/19/B/NZ6/00895). 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