DEHP induces immunosuppression through disturbing inflammatory factors and CYPs system homeostasis in common carp neutrophils

Fish and Shellfish Immunology 96 (2020) 26–31

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DEHP induces immunosuppression through disturbing inflammatory factors and CYPs system homeostasis in common carp neutrophils

T

Shuting Wanga, Yirong Caoa, Shengchen Wanga, Jingzeng Caia,∗∗, Ziwei Zhanga,b,∗ a b

College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, PR China Key Laboratory of the Provincial Education Department of Heilongjiang for Common Animal Disease Prevention and Treatment, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: DEHP Immuneresponses Common carp Cytochrome P450 Cytokines

Di-(2-ethylhexyl) phthalate (DEHP), a common pollutant in the water environment, has been reported to be associated with immune functions, especially aquatic organisms. However, whether DEHP exposure causes neutrophils toxicity in common carp is still unclear. To investigate the toxic effect of DEHP on immune functions, common carp neutrophils were exposed to DEHP (40 μmol/L and 200 μmol/L) for 2 h. The common carp neutrophils exposed to DEHP showed a decrease in neutrophil phagocytosis rate compared with control group. DEHP exposure induced a significant decrease in mRNA expression levels of inflammatory cytokines-related genes (Interleukin-6, Interleukin-8, transforming growth factor, tumor necrosis factor (TNF)-α, TNF-R1, TNF-T1, Interferon (IFN)-2a, IFN-g2b, IFN-g1) in common carp neutrophils, while the expression levels of IL-1β and IL-10 were increased compared with control group (P < 0.05). Furthermore, the detection of cytochrome P450 enzyme related genes showed that the mRNA expression levels of CYP (cytochrome P450 proteins)-1A, CYP-1B1, CYP-C1, CYP-2K were significantly decreased, and the mRNA expression level of CYP-3A was significantly reduced (P < 0.05). The results indicated that DEHP could affect the phagocytic ability of neutrophils by regulating the expression of inflammatory cytokines and disrupting cytochrome P450 homeostasis, which caused the immunosuppression in common carp.

1. Introduction As a common plasticizer, Di-(2-ethylhexyl) Phthalate (DEHP) was wildly used in plastic products including paint, food packaging, medical devices, toys and child-care products. Several studies have revealed that DEHP could cause pathological change and damage in the cardiovascular system [1,2]. However, DEHP is one of the most widespread environmental contaminants in the water environment [3], because it is easy to release into the environment once it encounters water or organic solvents. DEHP was reported to reach a level of 45.73 μg/g in the Yellow River, and 1.56 μg/g (dry weight) in the Qiantang river sediment in China [4]. It can enter the organism through inhalation, ingestion or dermal exposure, producing toxic effects on health [5,6]. Recent researches indicated that DEHP could affect the tissue development, endocrine system and antioxidant system of mammal and aquatic animals [7–10]. Fish species can be considered as top consumers in aquatic ecosystems [11]. Therefore, fish is widely used in the assessment of aquatic ecosystems health, and some biochemical changes in fish are considered as biomarkers of environmental pollution

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[12]. Micropollutants present in the aquatic environment may accumulate in fish and create a potential risk, not only to the fish themselves but also to piscivorous animals, including humans [13]. Common carp is an important fish for freshwater aquaculture in China. It is also widely distributed around the world and thence often used in toxicology research as a representative fish. Despite some efforts in studying the toxicity of DEHP, relatively little consideration has been given to the molecular mechanism of immune response induced by DEHP in common carp. Many studies have shown the effect of DEHP on the immune response. It has reported that DEPH could cause immunosuppression in organisms in different ways [14,15]. In addition, the toxic effects of many other micro-pollutants on the immune response may result in bodily death. Besides, the toxic effects of many other micropollutants on the immune response might result in decreasing the anti-infection of the immunological cell, or a significant increase in innate immune parameters of organisms [16,17]. Neutrophils are a very important part of innate immunity, which could phagocytize and destroy bacteria, release cytokines and activate elements of the innate immune response

Corresponding author. College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, PR China. Corresponding author. E-mail addresses: [email protected] (J. Cai), [email protected] (Z. Zhang).

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https://doi.org/10.1016/j.fsi.2019.11.073 Received 21 October 2019; Received in revised form 18 November 2019; Accepted 30 November 2019 Available online 30 November 2019 1050-4648/ © 2019 Elsevier Ltd. All rights reserved.

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[18,19]. These cells are capable of modulating components of adaptive immunity [20]and are very sensitive to water pollution in fish [18]. Phagocytosis of neutrophils could directly reflect the level of immune function. Many inflammation-related cytokines, such as interleukins (IL), tumor necrosis factor (TNF), and interferon (IFN), play a regulatory role in the immune response. IL-6, IL-8, TNF-α, and IFN-γ are important pro-inflammatory factors, whereas IL-10 inhibits inflammation in various diseases. These inflammatory factors can be detected in the expression products of neutrophils and could be used as indicators for determining their immune function. The cytochrome P450 isoenzyme (CYPs) is a hemoglobin superfamily, it is a terminal oxidase of the mixed-function oxidase system on the endoplasmic reticulum membrane. CYPs play a crucial role in the metabolisms of many detrimental substances including drugs, xenobiotics and herbicides [21,22]. The CYP1 subfamily is the most widely studied CYPs form in fish, and it is mainly involved in the metabolism of polycyclic aromatic compounds and aromatic amines. The CYP3 family is involved in the metabolism of a large number of endogenous and exogenous compounds. It can be induced by steroids, macrolide antibiotics, antifungals and phenobarbital. It was reported that in human hepatocytes in primary culture, the mRNA expressions of CYPs (CYP1A1, CYP1A2, and CYP3A4) were potent with cytokines (IL-6, ILlα, and TNF-α) [23]. More physiological and pathological effects of CYPs have been revealed by many researchers in recent years. In the present study, we used contrast experiments to explore the effects of DEHP on the immune response of common carp neutrophils. The results may provide a theoretical basis for preventing and resolving damage to fish organisms caused by plasticizer contamination.

Table 1 Gene-target primers used in qRT-PCR. Target gene

Primer Sequence (5' - 3′)

TNF-α

Forward 5′-GCTGTCTGCTTCACGCTCAA-3′ Reverse 5′-CTTGGAAGTGACATTTGCTTTT-3′ Forward 5′-TGTGCTGGACGAGGTGCCTATC -3′ Reverse 5′-CTTGGAAGTGACATTTGCTTTT-3′ Forward 5′-ATGAGCAGAAATCGAGGGCTCTC -3′ Reverse 5′-ACAGCCCTCAGAGGAGCCCTT-3′ Forward 5′-TCTTCACATTCTTCCGAGTT-3′ Reverse 5′-CCGAGGTAGTCCAGTTCT-3′ Forward 5′-AGCAGCGGGTGGAGGATGTA-3′ Reverse 5′-CCTCAGAAATGGCGGTGGAC-3′ Forward 5′-GACCTTCGCCCTCCACAG-3′ Reverse 5′-CCTCATCCCGATGCATGTTC-3′ Forward 5′-CAGGACGCAGGGTTTGTCTTGG-3′ Reverse 5′-GGTGGAGTTGAGAAGCGGTAGC-3′ Forward 5′-CTGGTCACGTCAGGGGTAGGC-3′ Reverse 5′-CCGAGGGCTCAACAGTTGCTG-3′ Forward 5′-ATGTCGTGTGTGATGAGCGG -3′ Reverse 5′-ATGACGTCGGCTTCCTCCA-3′ Forward 5′-TGCAGTGTTACCTGGGAGAA -3′ Reverse 5′-CGGTGTGATTTAGACCCGTAA -3′ Forward 5′-CCTGGCCCTCCTCCTGGTTTC-3′ Reverse 5-CACTGGCACCGCAGCTCATTC-3′ Forward 5′-CAGCACCAGTCATCAGCAGAGC-3′ Reverse 5′-GCAGGTGAAGAAGCGGTGACAG-3′ Forward 5′-CGCTGCTGCTTGATAGAA-3′ Reverse 5′-ACTGAGTGAATGTCCTGAAG-3′ Forward 5′-TGAATCTTGAGGAACCTGAG-3′ Reverse 5′-AACTGTGTTGCTTCTCTGT-3′ Forward 5′-CAACAGCAGCACCAGCAAACTG-3′ Reverse 5′-TTATCGCCTTGCGTTGGACCTG-3′ Forward 5′-GAGGTGGAGGCTAACAAC-3′ Reverse 5′-GATGTGAGGAGAGGATTCG-3′

TNFR1 CYP1A1 CYP1B1 CYP1C CYP3A138 CYP2K TGF il-1β IL-6 IL-8 IL-10 IFN-g1 IFN-g2a INF-g2b β-actin

2. Materials and methods 2.1. Ethics statement and experimental animals

for 40 min. Treated cells were stained with Wright's method [24]. The morphological of neutrophils was observed by using Oil lens.

All procedures applied in this research were authorized by the Institutional Animal Care and Use Committee of the Northeast Agricultural University. Twenty common carps (Cyprinus common carpio L., mean body weight of 115 ± 10 g; mean body length of 15. 5 ± 1. 49 cm) were purchased from the local fish farm that specializes in freshwater fish species. The species were individually acclimated for a week in a 200-L stock tank at 21 °C programmed with a 12 h light/ dark cycle prior to avoid unnecessary interferences. Circulating tap water was taken from a local water supply company, and food was provided twice a day, and aeration was uninterrupted.

2.4. RNA extraction and quantitative real-time PCR (qRT-PCR) Total RNA was isolated from neutrophils in all groups using the TRIzol reagent. The reverse transcription of mRNA was performed by using the First-Strand cDNA Synthesis Kit (Tiangen Biotech Co. Ltd., Beijing) and qRT-PCR was carried out using the LightCycler® 480 II Detection System (Roche, Switzerland). Different genes involved in immune responses were chosen for validation using qRT-PCR, including interleukin (IL-1β, IL-6, IL-8, IL-10), interferon-γ (INF-γ), TNF-α, transforming growth factor (TGF), and primers of gene qRT-PCR for detoxification and stress (CYP1A1, CYP1B1, CYP1C, CYP2K and CYP3A138), along with housekeeping gene β-actin were shown in Table 1. Data were analyzed according to the 2−ΔΔCt method. The results were expressed as the relative mRNA levels.

2.2. Cell isolation and DEHP exposure The peripheral heparinized blood (20 ml) was collected by puncturing the caudal vein from 5 common carps (n = 5). The neutrophils were obtained from a fish peripheral blood by using the neutrophil separation kit (Beijing Solarbio Biological Manufacture CO., China). The cells were grown in Roswell Park Memorial Institute (RPMI)-1640 with 10% fetal bovine serum and diluted to a density of 1 × 106 cells/ ml. The neutrophils were treated with 40 μmol/L and 200 μmol/L DEHP for 2 h at 25 °C and 5% CO2, while neutrophils not treated with DEHP were used as a control group (5 replicates were collected independently in each experiment, 3 used for experimental verification).

2.5. Statistical analysis Data were collected from at least three independent experiments and all results are expressed as the means ± standard deviations. Statistical analyses were performed using GraphPad Prism 5.0 software, and all data were assessed using the t-test. Values of P < 0.05 were considered to denote significant differences.

2.3. Phagocytosis test of neutrophils

3. Results

The Escherichia coli (E. coli) strain was added to the sterile LB (LuriaBertani) medium (10 ml) at a ratio of 1:100 and placed in a constant temperature shaking incubator for 12 h (37 °C, 200 rpm). The concentration of E. coli and neutrophils was counted by a hemocytometer under the microscope. After 2 h of DEHP exposure, the medium was removed and the cells were washed with phosphate buffer saline (PBS) and then added RPMI-1640 mixed with E. coli (bacteria: cells = 10:1)

3.1. DEHP inhibited phagocytosis of neutrophils Neutrophils are capable of phagocytizing and destroying bacteria, which could be considered as a criterion for determining immune function. Thus, the phagocytosis of neutrophils was observed by using a 27

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Fig. 1. The effects of DEHP on the phagocytic function of common carp neutrophils. Neutrophils were treated by DEHP for 2 h. A: Neutrophils in control group phagocytose E. coli (a, b). B: In the presence of DEHP (40 μmol/L), the amount of E. coli phagocytosed by neutrophils was showed (c, d). And lysed neutrophils could also be seen in this group (c). C: The phagocytosis of neutrophils treated with 200 μmol/L DEHP was showed (e, f).

ubiquitous pollution of mother phthalate in the environment and the metabolites of DEHP could accumulate in humans. Recent researches have revealed that DEHP could induce heart ischemia [25], and the high content of Mono(2-ethylhexyl) Phthalate caused cardiotoxicity in humans, which are associated with increased cell proliferation and inhibition of apoptosis [26]. DEHP could affect the synthesis and metabolism of hormones [27,28], causing genital organ damage, including testicular and uterus [29–31]. Many other studies have also revealed the toxic effects of DEHP on the liver [32], respiratory [33] and nervous systems [34,35]. Many studies have shown that DEHP caused acute toxicity to aquatic organisms, and it will cause chronic toxicity to its growth and reproduction, resulting in decreased body condition and reproductive capacity [36–38]. DEHP exposure reduced the body weight of adult fish and their offspring [28]. DEHP exerts toxic effects to induce immunosuppression by interfering with hepatic metabolism [14]. The survival of the cultured immune tissue cells of rainbow trout exposed to DEHP was reduced with the increasing concentrations of DEHP exposure [39–41]. The exposure to DEHP could induce immunosuppressive mechanisms of the prophenoloxidase (proPO) system [15]. After exposure to DEHP, the inhibition of immune-related gene expression was present in clam [42,43]. In our study, neutrophils in the DEHP-treated group showed weaker phagocytic ability than the control group, and the higher concentration of DEHP, the lower the phagocytosis ability of the cells, which lead to the immunosuppressive effect of common carp. Thus, to further determine the immunosuppressive effect of DEHP to neutrophils, the mRNA expression levels of cytokines in immunity were detected. TNF-α, interleukin and IFN-γ are usually used to respond to the body's inflammatory status. TNF-α, IL-1β, IL-6, and IL-8 are classic proinflammatory molecules [44,45], while TGF-β and IL-10 are considered as anti-inflammatory cytokines [46,47]. Polychlorinated biphenyls (PCBs) have endocrine disruption properties to modulate immune responses by interfering with the mRNA levels of CYP1A [48]. IL-1β, IL-6 and INF-γ play important roles in fish immune response [49,50]. Abramson's study showed that low expression of IL-1β leads to abnormal activation of neutrophils, leading to the widespread release of pro-inflammatory mediators [51]. Large production of IL-1β increased endocrine effects: it induced acute phase protein synthesis in the liver and caused fever and cachexia [52,53]. The contents of inflammatory mediators, intercellular adhesion molecular (ICAM)-1 and IL-1β upregulation are possible early contributors to chronic obstructive pulmonary disease (COPD)-associated inflammatory lung injury [54]. IL10 attracts neutrophils and causes the release of inflammatory mediators. IL-10 was also considered as an inflammatory marker and a multifunctional cytokine with multiple roles in the regulation of inflammation/immune response. Previous studies had shown that IL-10 expression elevated in inflammation without exception. IL-10, that served as a chemoattractant for neutrophil, will increase the level of chemokine and also act on macrophages to enhance phagocytosis as

microscope (Fig. 1). Compared with the control group, the phagocytosis of E. coli by neutrophils in the DEHP group was significantly attenuated, accompanied by a large reduction in bacteria swallowed by neutrophils. In addition, the phagocytosis of neutrophils treated with DEHP was obviously rarer in high concentration group (200 μmol/L) than that in low concentration group (40 μmol/L) (Fig. 1A). The phagocytosis rates of the DEHP group were 29.09% (40 μmol/L) and 19.72% (200 μmol/L), evidently lower than that in the control group (56.25%) (Fig. 1B). The results indicated that the amount of E. coli phagocytosed by neutrophils was significantly downregulated in the presence of DEHP compared to the control group, and this kind of inhibition was positively correlated with the concentration of DEHP, which prompted the inhibition of phagocytic ability. 3.2. DEHP interfered with the expression of cytokines in neutrophils To verify the effect of DEHP on the immune function of neutrophil in common carps, several inflammatory cytokines (interleukin, interferonγ, TNF-α and TGF) were detected. The effect of DEHP exposure on the mRNA abundance of inflammatory cytokines-related genes (IL-6, IL-8, TNF-α, IFN-2a, IFN-g1, IFN-g2b, IL-10 and IL-1β) in common carp neutrophils was shown in Fig. 2. The qRT-PCR results revealed that the mRNA expressions of IL-6, IL-8, TNF-α, IFN-2a, IFN-g1, IFN-g2b in the DEHP group were decreased significantly (P < 0.05) compared to that in the control group (Fig. 2A), however, the expressions of IL-10 and IL-1β mRNA were increased (P < 0.05) in the DEHP group (Fig. 2B). The trends were changed in a dose-dependent way. 3.3. DEHP affected the CYPs system homeostasis in neutrophils The effect of DEHP exposure on the mRNA levels of CYPs-related genes CYP1 (CYP1A1, CYP1B1 and CYP1C1), CYP2K and CYP3A in neutrophils were shown in Fig. 3. The qRT-PCR results revealed that the mRNA expression levels of CYP1A1, CYP1B1, CYP1C1 and CYP2K were decreased in DEHP group, while the mRNA level of CYP3A was increased compared with the control group. 4. Discussion In the present study, by establishing a DEHP model of common carp neutrophils in vitro, the mRNA levels of cytokines in immunity and CYPs pathway-related genes were detected in neutrophils of common carp induced by DEHP. We indicated that DEHP could affect the phagocytic function of common carp neutrophils through inflammatory factors and CYPs pathways, ultimately, triggering the immunosuppression of immunocytes. DEHP is one of the most widely used phthalates caused by the 28

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Fig. 2. The effects of DEHP exposure on the mRNA abundance of inflammatory cytokines-related genes. A: The qRT-PCR results revealed that the mRNA expressions of IL-6, IL-8, IFN-γ and TNF-α were showed. B: The mRNA expressions of IL-1β and IL-10 were showed. The data was presented as the mean ± SD (n = 5). Samples with different letters were considered as significant differences (P < 0.05). The samples with a common letter were not significantly different (P > 0.05).

after exposure to DEHP compared to other groups. These abnormal expressions of cytokines suggested that the interleukin family factor was involved in the development of inflammation. Our research was consistent with the above, indicating that the increase of IL-1β, IL-4, and IL-6 and the decrease of IFN-g could occur under the treatment of

well as the production of other pro-inflammatory cytokines [55]. In addition, it enhanced the release of anti-inflammatory factors such as IL-1 receptor antagonists. Thus, IL-10 dramatically reduced the effects of important cytokines in most innate immunity. Similarly, our results showed that the expression of IL-10 was also increased significantly

Fig. 3. Effects of DEHP on neutrophil CYPs pathways. The qRT-PCR results revealed that in DEHP group, the mRNA expressions of CYP1A1, CYP1B1, CYP1C1, CYP2K and CYP3A were showed. The data were presented as the mean ± SD (n = 5). Samples with different letters were considered as significant differences (P < 0.05). The samples with a common letter were not significantly different (P > 0.05). 29

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LB E. coli qRT-PCR PBS proPO PCBs ICAM COPD ATR CPF

DEHP, and this imbalance reaction was strongly amplified with the enhancement of DEHP, suggesting that the exposure of DEHP inhibited the phagocytic ability of neutrophils by regulating the expression of inflammatory cytokines. Many toxic substances in aquatic environment could cause damage to the immune function of carp, and we have previously proved that chlorpyrifos and heavy metal cadmium could destroy the normal immune function of carp by affecting cytokines to lead to the death of fish and other kinds of aquatic animals eventually [56–58], whether DEHP could affect the function of neutrophils of common carp by affecting cytokines has not been reported yet. Our results showed that DEHP had an influence on the immune response of neutrophils by interfering with the expression of cytokines, which could cause an immunosuppressive effect in common carp. The CYP1 subfamily is the most widely studied CYPs form in fish and has been considered the most sensitive RNA biomarker for evaluating environmental pollution. Polychlorinated biphenyls (PCBs) have endocrine disruption properties to modulate immune responses by interfering with the mRNA levels of CYP1A and P4501A [59,60]. The mRNA expressions of CYPs (CYP1A1, CYP1A2, and CYP3A4) were potent with cytokines (IL-6, IL-lα, and TNF-α) in human hepatocytes in primary culture. Multiple studies have reported that the cellular apoptosis level was related to the observation of CYP450 activity [2]. Previous studies showed that the combined exposure of Atrazine (ATR) and Chlorpyrifos (CPF) significantly increased mRNA levels of CYP1 (CYP1A, CYP1B and CYP1C) in common carp gills [61,62]. Recently, our previous studies have shown that DEHP exposure caused oxidative stress and resulted in apoptotic injury through the mitochondrial pathway and the cytochrome P450 (CYP) pathway [2,63]. In this study, we found that CYP1A, CYP1B and CYP1C expressions were downregulated in common carp neutrophils after DEHP treatment, and we speculated that DEHP may interfere the metabolism of poisons in neutrophils by inhibiting the transcription of CYP1. CYP3A was involved in drug metabolism and its cloned gene in common carp was identified as CYP3A138. Researches showed that paraquat exposure could spur the transcription of CYP3A138 in the livers of common carp. In our research, the expression of CYP3A138 mRNA was enhanced in DEHP-treated groups, which indicated that CYP3A may take part in the metabolism of DEHP in neutrophils of common carp. In conclusion, among the immune cells, DEHP exposure could induce immunosuppressive effects of neutrophils by restraining the expression of cytokines and disturbing the CYPs system homeostasis in neutrophils. Thus, our results provide a more enhanced understanding of the interplay between DEHP exposure and immune responses.

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Declaration of competing interest The authors declare that there is no conflict of interest. Acknowledgments This work was supported by the National Project of Students Innovative Training (SIPT) program of Northeast Agricultural University in 2019 (No.201910224020 and No.201910224001). Abbreviations DEHP IL-1β IL-6 IL-8 IL-10 CYP TGF TNF-α IFN-γ RPMI

Luria-Bertani Escherichia coli Quantitative real time polymerase chain reaction phosphate buffer saline Phenol Oxidase Activation Polychlorinated biphenyls intercellular adhesion molecular chronic obstructive pulmonary disease Atrazine Chlorpyrifos

Di-(2-ethylhexyl) phthalate interleukin 1β interleukin 6 interleukin 8 interleukin 10 cytochrome P450 transforming growth factor tumor necrosis factor-α Interferonγ Roswell Park Memorial Institute 30

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