Aflatoxin B1 Suppressed T-Cell Response to Anti-pig-CD3

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Aflatoxin B1 Suppressed T‑Cell Response to Anti-pig-CD3 Monoclonal Antibody Stimulation in Primary Porcine Splenocytes: A Role for the Extracellular Regulated Protein Kinase (ERK1/2) MAPK Signaling Pathway Shu Hao, Shengchi Pan, Junfa Hu, Gang Qian, Fang Gan, and Kehe Huang* Institute of Nutritional and Metabolic Disorders in Domestic Animals and Fowls, Nanjing Agricultural University, Nanjing 210095, China ABSTRACT: The aim of the present study is to investigate whether aflatoxin B1 (AFB1)-induced immunotoxicity is associated with oxidative stress and the expression of extracellular regulated protein kinases (ERK) 1/2. The primary splenocytes isolated from healthy pigs were activated and proliferated by anti-pig-CD3 monoclonal antibodies (mAb) in the present experiment, which is an antigen-specific stimulant. Results indicated that cell proliferation and interleukin-2 (IL-2) production were significantly suppressed by AFB1 from 4 to 8 μg/mL in a dose-dependent manner compared to the control group. Furthermore, AFB1 significantly increased malondialdehyde (MDA) levels, decreased reduced glutathione (GSH) and total superoxide dismutase levels, and up-regulated p-ERK1/2 expression in the activated splenocytes. N-Acetyl-L-cysteine blocked anti-CD3induced T-cell suppression by AFB1 through increasing intracellular concentrations of GSH levels, decreasing MDA levels, and down-regulated p-ERK1/2 expression, respectively. Inhibition of the ERK1/2 expression by ERK-specific iRNA attenuated the decrease of T-cell proliferation and IL-2 production induced by AFB1. It was concluded that AFB1 inhibits anti-CD3-induced lymphocyte proliferation and IL-2 production by the oxidative stress mediated ERK1/2 MAPK signaling pathway. KEYWORDS: aflatoxin B1, primary porcine splenocytes, anti-pig-CD3 mAb, RNA interference, ERK1/2

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INTRODUCTION Aflatoxin B1 (AFB1), a mycotoxin produced by Aspergillus flavus and Aspergillus parasiticus, presents potential hepatotoxic, carcinogenic, and immunotoxic properties and threatens human and animal health safety.1−3 Among the numerous types of aflatoxins identified, AFB1 is the predominant form and is considered as extremely toxic.4 Extreme AFB1 doses exhibit acute poisoning and death in humans and animals. For example, an outbreak of acute aflatoxicosis in Kenya was one of the most severe episodes of human aflatoxin poisoning in history. A total of 317 cases were reported with a case fatality rate of 39%.5 Occurrence of acute aflatoxicosis in animals is more common, which is characterized by abdominal pain, pulmonary or cerebral edema, necrosis, and fatty liver.6 However, low-dose exposure is also an interesting research topic because chronic toxin poisoning is associated with weight loss and poor performance, brings change in clinical biochemistry patterns, suppresses immune function, increases susceptibility to infectious diseases and mortality, and causes potential economic losses in livestock husbandry.7 Various animal studies and in vitro experiments have demonstrated that AFB1 mainly exerts its effects on cellmediated immunity, because humoral immune suppression is less consistent between different species and seems to require higher doses of aflatoxins.8 Ingestion of contaminated feeds increases susceptibility to infection and decreases protection conferred by vaccination in pigs.8 Mehrzad et al.9 indicated that a low level of AFB1 deregulated the antigen-presenting capacity of porcine dendritic cells. Analysis of blood lymphocytes from pigs fed AFB1-contaminated diet and stimulated in vitro with © XXXX American Chemical Society

mitogen revealed a decreased expression of IL-1β and increased expression of IL-10.10 However, the mechanism and pathway remain largely unclear, and some results of pig experiments are somewhat conflicting. Pigs are extremely sensitive to AFB1,11 and exposure to this toxin induces enormous economic losses in China.12−14 Otherwise, because of the great physiological similarities between humans and pigs, such as food intake, energy expenditure for body size, body proportion, and innate or adaptive immune system,15−18 the pig is considered as a tractable model for human immunity.19 T lymphocyte activation and proliferation are important evaluations for cellular immune function, and T lymphocyte stimulation is used extensively to facilitate T-cell expansion and in the study of T-cell function in vitro.20 Interleukin-2 (IL-2) production is a hallmark of activated T cells.21 T-cell receptor (TCR) is expressed on mature T cells specifically to recognize non-self-antigens, which are involved in T-cell development, survival, and activation and defend the host from infectious diseases.22 Stimulation of monoclonal antibodies (mAb) is proved to be more specific than mitogen response in some studies.23 In many studies on TCR signaling, mAb that recognize the invariant ectodomain of CD3ε have been used to activate T cells.24 Considerable evidence supports the contention that oxidative stress is one of the basic factors in the etiology of numerous Received: January 23, 2015 Revised: June 13, 2015 Accepted: June 13, 2015

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To assess T-cell proliferation, 1 × 106 cells/well CFSE-stained splenocytes were cultivated in 24-well plates accompanied by RPMI1640 medium to a final volume of 1 mL. After 60 h, splenocytes were centrifuged and wash with phosphate buffered saline (PBS) once, then stained with anti-CD3-PEcy5 (Abcam, UK) and prepared for flow cytometry analysis. Flow cytometry was used and enabled by FACSCalibur (BD Biosciences, USA). Color compensation was achieved using an appropriate single fluorochrome-labeled sample. Data were analyzed using FlowJo 7.6.5 (TreeStar). Determination of IL-2 in Splenocytes. Splenocytes (1 × 106 cells/well) were seeded in 96-well plates and treated with various concentrations of AFB1 (0, 1, 2, 4, and 8 μg/mL) in the absence (controls) or presence of anti-pig-CD3 mAb for 60 h. The cell supernatants were then collected and assayed for IL-2 by ELISA (R&D Systems, USA). All samples were assayed in triplicate. Oxidative Stress Analysis. Primary porcine splenocytes at a density of 2 × 106/well in 6-well plates were treated with AFB1 at 0, 1, 2, 4, and 8 μg/mL for 60 h in the presence of anti-pig-CD3 mAb. Intracellular glutathione (GSH) in splenocyte cytosol was measured as previously described,32 using commercially available kits (Jiancheng, China), which are based on the reaction of 5,5′-dithiobis(2nitrobenzoic acid) and GSH to 5-thio-2-nitrobenzoic acid. Cell extracts were prepared, and GSH was measured spectrophotometrically (412 nm). Protein concentration was determined according to the BCA method using commercially available kits (Beyotime, Jiangsu, China). Data were expressed as micromoles of GSH per gram of protein. All samples were assayed in triplicate. Assessment of T-SOD enzymatic activity was performed according to a previously described method,33 using commercially available kits (Jiancheng, China). The absorbance was measured at 525 nm for 10 min. One unit of T-SOD was defined as the enzyme amount responsible for 50% of the inhibition of epinephrine oxidation. T-SOD enzymatic activity was expressed as units (U) per milligram of protein. All samples were assayed in triplicate. Intracellular malondialdehyde (MDA) in splenocyte cytosol was measured as previously described,34 using commercially available kits (Jiancheng, China). All samples were assayed in triplicate. Western Blot Analysis. Spleen lymphocyte suspension was cultured in a 6-well plate at a density of 2 × 106/well for 60 h with a density of 2 × 106 cells/well. After separation by SDS-PAGE, protein molecules on the gel were transferred onto a PVDF membrane and then probed using a specific antibody to quantitatively detect ERK (1/ 1000) (Cell Signaling Technology, USA) and p-ERK (1/2000) protein (Cell Signaling Technology, USA). After the first antibody reaction overnight, the PVDF membrane was washed by TBST three times and incubated for 60 min with horseradish peroxidase (HRP)labeled anti-rabbit secondary antibody (Cell Signaling Technology, USA), diluted 1/5000 (v/v) in 5% (w/v) BSA TBST. Finally, the result of Western blot was detected by electrochemiluminescence (ECL) using the Amersham Hyperfilm ECL reagent. Short Interfering RNA (siRNA) Transfection. Double-stranded RNA sequences were designed according to the sequence of ERKspecific siRNAs using Invitrogen BlockiT RNAi designer and were synthesized by Invitrogen. The ERK-specific siRNAs sequence was sense 5′-CUCCAAAGCUCUGGAUUUAtt-3′ and antisense 5′-UAAAUCCAGAGCUUUGGAGtt-3′. The control siRNA had the sequence 5′-UUCUCCGAACGUGUCACGUTT-3′. Duplexes were resuspended in DEPC-treated water to obtain 20 mM solutions before use. T cells in RPMI-1640 medium without FBS or antibiotics were seeded in 6-well plates, 24-well plates, and 96-well plates after spleen lymphocytes were separated, and siRNA was introduced using the XtremeGene siRNA transfection reagent (Roche, USA) according to the manufacturer’s protocol. Statistical Analysis. All of the data were analyzed statistically using the SPSS 18.0 for Windows statistical software package (Statistical Product and Service Solutions, Inc., USA). All results are presented as the mean ± SEM. We also performed a one-way ANOVA on the statistical analysis. Tukey’s b (K) post-test was used to compare means of each group, and a P value was considered significant at 0.05.

diseases, and oxidative stress has been shown to activate extracellular signal-regulated kinases (ERK) 1/2 simultaneously.25,26 Interestingly, previous results also show that antioxidants (e.g., N-acetyl-L-cysteine, NAC) added before other toxin treatment can rescue cells from cytotoxicity induced by the toxin through decreased oxidative damage.27 In addition, the ERK signaling pathway is one of the major pathways of T lymphocyte proliferation and IL-2 production.28−30 Until now, the interaction of AFB1-induced immunotoxicity and the ERK1/2 pathway in T lymphocyte proliferation is relatively unexplored. Because a lot of research shows AFB1 exposure could exert oxidative stress in cell culture model,31 we hypothesize that (i) AFB1 could induce immunotoxicity in primary porcine splenocytes and (ii) AFB1 induced decreases of porcine splenocyte proliferation and IL-2 production by oxidative stress mediated ERK1/2 MAPK signaling pathway. Thus, our objective was to evaluate the effect of different concentrations of AFB1 on the anti-CD3-induced primary porcine splenocytes proliferation and IL-2 production, intracellular redox status, and expression of p-ERK1/2 MARK protein and to determine whether intracellular oxidative stress and the phosphorylation of the ERK1/2 MARK pathway are novel mechanisms involved in AFB1-induced immunotoxic properties in primary porcine splenocytes.

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MATERIALS AND METHODS

Animal and Cell Culture. The use of experimental animals and the following procedures were approved by the Nanjing Agricultural University Animal Care Committee. Fifty normal Meishan pigs (half males and half females), aged 30 days and weighing 8 ± 2 kg, were collected from the breeding center of Jiangsu Polytechnic College of Agriculture and Forestry and killed. The spleens were removed and placed in Hanks’ solution and then disrupted mechanically under a clean bench, after which they were carefully layered on the surface of lymphocyte separation medium (Tianjin Hematology Institute, Chinese Academy of Medical Sciences, China). Isolated spleen lymphocytes were adjusted and supplemented with enriched RPMI1640 medium (Invitrogen, USA), which was added with 10% fetal bovine serum (FBS), penicillin (20 mg/mL), and streptomycin (20 mg/mL) and cultivated at 37 °C in the incubator with 5% CO2, and subsequently evaluated for the viability of isolated spleen lymphocyte by 0.4% trypan blue dye exclusion. We detected lymphocyte proliferation at 24, 36, 48, 60, and 72 h in the beginning stage of the experiment. However, we found that cell proliferation at 60 h had the best results by flow cytometry, so we detected cell proliferation only at 60 h in the following experiment. In previous literature, the antigen-specific response is more sensitive to AFB1 exposure than the mitogen-specific response in pigs, so we selected only anti-CD3 stimulation rather than mitogen stimulation in the present experiment. AFB1 (Sigma, USA) was dissolved in DMSO (1 mg/mL), and final mycotoxin concentrations were diluted in the culture medium. Cells without any AFB1 and with 0.4% final concentration of DMSO were used as the control group. Spleen Lymphocyte Proliferation Assay. Spleen lymphocytes (1 × 106 cells/well) were treated with various concentrations of AFB1 in the absence or presence of anti-pig-CD3 mAb (clone PPT3, Abcam, UK) and cultivated in 96-well plates for 60 h. In the splenocyte viability experiment, AFB1 was exposed at 0, 0.5, 1, 2, 4, 8, 10, and 20 μg/mL concentrations. In cell proliferation analysis, AFB1 was treated at 0, 1, 2, 4, and 8 μg/mL concentrations. Subsequently, 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (Sigma, USA) was added at 5 mg/mL concentration, and the plate was oscillated for 10 min by enzyme-labeled instrument (BioRad, USA). Finally, the optical density (OD) value was assessed as absorbance at 490 nm. All samples were assayed in quadruplicate. B

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Figure 1. Effects of AFB1 on the viability, cell proliferation, and IL-2 production in primary porcine splenocytes. Primary porcine splenocytes (1 × 106 cells/well) were treated with different concentrations of AFB1 for 60 h without any stimulus (A) or stimulated by 2 μg/mL anti-pig-CD3 mAb (B) and were assayed by the MTT method. The values are presented as means ± SEM (n = 4). IL-2 production in splenocytes treated with different concentrations of AFB1 and IL-2 concentrations was determined by ELISA (C). The values are presented as means ± SEM (n = 3). Significant differences with control were designated as ∗ P < 0.05 and ∗∗ P < 0.01. CD3+T-cell proliferation was monitored using CFSE labeling (D). Cells were stained with anti-CD3-PEcy5. CD3+T cells from the entire well were analyzed for proliferation by flow cytometry. The percentage of proliferating cells for each culture is indicated. A representative experiment from two separate experiments is shown.

Figure 2. Effects of AFB1 supplementation on oxidative stress capacity. Primary porcine splenocytes at a density of 2 × 106/well in 6-well plates were treated with AFB1 at 1, 2, 4, and 8 μg/mL for 60 h in the presence of anti-CD3. Changed levels of GSH (A), T-SOD (B), and MDA (C) were assayed as described under Materials and Methods. The values are presented as means ± SEM (n = 3). Significant differences with control were designated as ∗P < 0.05 and ∗∗ P < 0.01. Supplementary Information. Primary porcine splenocytes were stimulated with 2 μg/mL anti-pig-CD3 mAb in the absence or presence of 5 mmol/L of NAC for 24 h, washed with PBS once, and then treated with AFB1 (4 μg/mL) or not for another 36 h. Moreover, control and NAC treatment group were treated at the final concentration of 0.4% DMSO.

observation, the higher dose was excluded during our experiments. Over the range of concentrations of AFB1 used, the proliferation of cells was significantly lowered by AFB1 at 4 and 8 μg/mL in a dose-dependent manner (P < 0.05) (Figure 1B). A significant reduction in IL-2 production by anti-pig-CD3 stimulation was observed at concentrations of 4 and 8 μg/mL (P < 0.05). The inhibition indicates dose dependency (Figure 1C). To confirm whether the proliferation evaluated by cell counting assay was due to T lymphocytes or total cells or any other cell type, CD3+T-cell proliferation was estimated by CFSE labeling of splenocytes and following anti-pig-CD3PEcy5 staining. The T-cell proliferation measured by CFSE showed a pattern similar to that of MTT. Total CD3+T cells proliferated less in the toxin exposure group than in the control group with anti-pig-CD3 mAb regulated and treated (19.82 versus 42.77%, Figure 1D).

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RESULTS Effects of AFB1 on Viability, Cell Proliferation, and IL-2 Production in Primary Porcine Splenocytes. To investigate the effect of AFB1 treatment on the antigen-stimulated lymphocyte proliferation, we examined the effects of AFB1 at various concentrations on splenocyte proliferation and IL-2 production. As shown in Figure 1, DMSO, the solvent of AFB1, does not have a significant effect on splenocyte viability and proliferation of lymphocytes. However, the higher doses of AFB1 (10 and 20 μg/mL) showed significant cytotoxic effect on the viability (P < 0.05) (Figure 1A). On the basis of this C

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Figure 3. Effects of NAC on cell proliferation, IL-2, and redox activity induced by AFB1 in primary porcine splenocytes. Primary porcine splenocytes were stimulated with anti-CD3 in the absence or presence of 5 mmol/L of NAC for 24 h and then inoculated with or without AFB1 at 4 μg/mL for a further 36 h treatment. Cells were assayed for cell proliferation (A), IL-2 production (C), levels of GSH (D), T-SOD (E), and MDA (F). Data are presented as means ± SE of three independent experiments: ∗ P < 0.05 and ∗∗ P < 0.01 are statistically significant compared to control cells; # P < 0.05 and ## P < 0.01 are statistically significant compared to 4 μg/mL AFB1-treated T cells. CD3+T-cell proliferation was monitored using CFSE labeling (B). Cells were stained with anti-CD3-PEcy5. CD3+T cells from the entire well were analyzed for proliferation by flow cytometry. The percentage of proliferating cells for each culture is indicated. A representative experiment from two separate experiments is shown.

Figure 4. Western blot and densitometric analysis of ERK1/2 expression. Primary porcine splenocytes were treated with AFB1 at 1, 2, 4, and 8 μg/ mL for 60 h in the presence of anti-CD3 (A). Primary porcine splenocytes were stimulated with anti-CD3 in the absence or presence of 5 mmol/L of NAC for 24 h and then inoculated with or without AFB1 at 4 μg/mL for a further 36 h treatment (B). Whole cell ERK expression/β-actin was used as a control. Densitometric analysis results are expressed as the ratio of phosphorylated ERK to β-actin and the whole cell ERK expression to β-actin. Graphical results are represented the mean ± SEM of three independent experiments: ∗ P < 0.05 and ∗∗ P < 0.01 are tatistically significant compared to control cells; # P < 0.05 and ## P < 0.01 are statistically significant compared to 4 μg/mL AFB1-treated T cells.

Effects of AFB1 Supplementation on Oxidative Stress Capacity. To investigate the effect of AFB1 exposure on the

parameters of oxidative stress, GSH, T-SOD, and MDA levels were measured in the splenocytes. Cells treated with AFB1 D

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Figure 5. Effect of ERK-knockdown on ERK1/2 and p-ERK1/2 expression and oxidative stress in primary porcine splenocytes. Cell were transfected with either an ERK-specific siRNA or a control siRNA and cultured for 5 h before 4 μg/mL AFB1 and 2 μg/mL anti-CD3 treatment. Cell samples were assayed for p-ERK and ERK protein levels (A) and GSH (B), T-SOD (C), and MDA (D) levels as described under Materials and Methods. Whole cell ERK expression/β-actin was used as a control. Densitometric analysis results are expressed as the ratio of phosphorylated ERK to β-actin and the whole cell ERK expression to β-actin. Graphical results are represented the mean ± SEM of three independent experiments: ∗ and # indicate statistically significant difference from control (* and #, P < 0.05; ∗∗ and ##, P < 0.01).

decreasing MDA levels (Figure 3F), respectively, compared with the AFB1 group (P < 0.05). However, NAC together with AFB1 did not produce a significant increase in the IL-2 production and intracellular concentrations of T-SOD levels (Figure 3C,E). Western Blot and Densitometric Analysis of p-ERK1/2 and ERK1/2 Expression. To determine if the AFB1 treatment of primary porcine splenocytes affects expression of extracellular signal-regulated kinases, Western blot was used to measure protein levels of ERK1/2 protein in activated splenocytes. The significant change in p-ERK1/2 expression was associated with a dose-dependent increase in gray value. As shown in Figure 4A, in the cells exposed to various concentrations of AFB1 (1−8 μg/mL), AFB1 at a concentration of 2 μg/mL showed significantly higher ERK1/2 phosphorylation compared to the no-toxin group (P < 0.01). It has been assumed that the influence of AFB1 on cell proliferation shows connections with p-ERK1/2 protein expression in T lymphocytes. As shown in Figure 4B, expression of p-ERK1/ 2 protein in activated splenocytes was decreased in NAC interaction with the AFB1 group compared to the AFB1 group. Effect of ERK-Knockdown on ERK1/2 Expression and Oxidative Stress in Primary Porcine Splenocytes. The extent of ERK-knockdown was evaluated by determination of ERK1/2 protein levels after primary porcine splenocyte cells were transfected with either an ERK1/2-specific or a control siRNA. As shown in Figure 5, transfection of primary porcine splenocytes cells with ERK-specific siRNA led to a decrease in ERK1/2 protein levels compared with control cells, there being

showed a dose-dependent decrease in the intracellular concentrations of GSH. The minimal decreases of GSH levels appeared in activated splenocytes exposed to 8 μg/mL AFB1 (Figure 2A) and GSH concentrations in the 4 μg/mL AFB1 show a significant decrease compared with control splenocytes, whereas T-SOD presents the same trend (Figure 2B). To assess the MDA levels in primary porcine splenocytes, which is a marker of oxidative stress, we measured the results as shown in Figure 2C. In 0−8 μg/mL AFB1-treated groups, MDA concentrations exhibit a dose-dependent increase. MDA concentrations in the 1 μg/mL AFB1 show a significant enhancement compared with control splenocytes. The maximal increases of MDA level were observed in activated splenocytes incubated with 8 μg/mL AFB1. Effects of NAC on Cell Proliferation, IL-2, and Redox Activity Induced by AFB1 in Primary Porcine Splenocytes. To determine whether a change of oxidative stress could affect T-cell proliferation, primary porcine splenocytes were stimulated with 2 μg/mL anti-pig-CD3 mAb in the absence or presence of 5 mmol/L of NAC for 24 h. As shown in Figure 3A, AFB1 treatment exhibited significant toxicity, which was suppressed in NAC-treated cells (P < 0.05). The T-cell proliferation measured by CFSE showed a pattern similar to that of MTT (Figure 3B). Total CD3+T cells proliferated more in the NAC exposure group and the NAC and AFB1 group than in the AFB1 group with anti-pig-CD3 mAb treatment (58.22 and 31.96 versus 19.82%). We found that NAC alleviated AFB1-induced cytotoxicity as demonstrated by increasing intracellular concentrations of GSH levels (Figure 3D) and E

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Figure 6. Alleviative effects of ERK1/2 knockdown on immunosuppression induced by AFB1 in primary porcine splenocytes. Cells were transfected with either an ERK-specific siRNA or a control siRNA and cultured for 5 h before 4 μg/mL AFB1 and 2 μg/mL anti-CD3 treatment. The transfected cells were assayed for proliferation by MTT (A) and IL-2 concentration by ELISA (C). Cells with only stimulus were used as control. Data are presented as means ± SE of three independent experiments: ∗ P < 0.05, statistically significant compared to control cells; # P < 0.05, statistically significant compared to 4 μg/mL AFB1-treated T cells. CD3+T-cell proliferation was monitored using CFSE labeling (B). Cells were stained with anti-CD3-PEcy5. CD3+T cells from the entire well were analyzed for proliferation by flow cytometry. The percentage of proliferating cells for each culture is indicated. A representative experiment from two separate experiments is shown.

induced by AFB1 have not been reported in pigs until now. The present work is the first to investigate the relationship of cell signaling pathway and the immunotoxicity induced by AFB1. The present data showed that the effects of AFB1-induced immunotoxicity were associated with oxidative damage and the ERK1/2 MAPK signaling pathway. Costello et al. demonstrated an overall higher activation status achieved with CD3i/CD28i than with PHA. 37 Meissonnier et al.8 supplied results that the antigen-specific response is more sensitive to AFB1 than the mitogen-specific response in blood lymphocytes exposed to dietary AFB1 from pigs. Therefore, we chose anti-CD3 stimulation rather than mitogen stimulation in the present experiment. Lymphocyte proliferation and IL-2 production were considered as measures to evaluate T-cell function in an in vitro experiment.38 Our results showed that the vitality of AFB1-induced unstimulated T cells was decreased dose dependently, but relatively insensitively, compared to the active T cells, which was consistent with a previous study that AFB 1 decreases lymphoid cell populations, especially activated lymphocytes.8 In anti-CD3induced T cells, 4−8 μg/mL AFB1 exposures significantly inhibited T-cell proliferation and IL-2 production in the present work. Actually, concentrations of >8 μg/mL had been detected and had a continued declining trend (data not shown). To ensure the accuracy of the results, both MTT and flow cytometry were used as effective methods to measure the proliferation of T cells in the present experiment. To our knowledge, reactive oxygen species (ROS) not only result from normal cellular metabolism but also are related to

a 65% decrease in p-ERK1/2 protein levels and a 55% decrease in whole cell ERK1/2, respectively (Figure 5A). Transfection of primary porcine splenocytes with siRNA targeted against ERK had no effects on the redox status as measured by intracellular GSH levels, T-SOD levels, and MDA levels (Figure 5B−D). Alleviative Effects of ERK-Knockdown on Immunosuppression Induced by AFB1 in Primary Porcine Splenocytes. To address whether ERK1/2 plays a key role in reducing the cytotoxicity of AFB1 in primary porcine splenocytes, cells were cultured and then transfected with ERKspecific or control siRNA. After 5 h of transfection treatment, the medium was removed. Transfected cells were then cultured with AFB1 at 4 μg/mL in the presence of anti-pig-CD3 mAb for a further 55 h. As shown in Figure 6, ERK-specific siRNA alleviated the immune toxicity of AFB1, as demonstrated by increasing T-cell proliferation (Figure 6A) and IL-2 production (Figure 6C) (P < 0.05), but not in cells transfected with the siRNA control. The T-cell proliferation measured by CFSE showed a pattern similar to that of MTT (Figure 6B). Total CD3+T cells proliferated more in the ERK-specific siRNA group than in the AFB1 exposure group and control siRNA group with anti-pig-CD3 mAb treatment (33.75 versus 19.82 and 21.02%).

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DISCUSSION Previous studies have indicated that an AFB1-contaminated diet has been widely documented in many parts of the world and exposure to the toxin affects the immune response in pigs.35,36 However, the detailed mechanisms of the immunotoxicity F

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the treatment of the drugs or environment pollutants.39 Parveen et al. showed that 0.25 μg/mL AFB1 caused a significant increase in oxidative stress in MDCK cells, which was demonstrated by a significant increase of MDA level and reduction of intracellular GSH level, as well as GPX1 activity and mRNA level.40 Our results showed that AFB1 treatment induced intracellular oxidative stress in the splenocyte, which is consistent with previous studies.41 The results from the NAC treatment experiments provided further evidence that AFB1-induced T-cell inhibition is associated with cellular oxidative stress in splenocytes. NAC is a pharmacological antioxidant. Treatment of NAC in splenocytes reduced oxidative stress and resulted in an alleviation of T-cell proliferation. Xiao et al. confirmed that the significant toxicity induced by 4-ClBQ could be suppressed after NAC treatment in human keratinocytes.27 Our study showed that NAC alleviated the immunotoxic effects of AFB1, which indicated that the AFB1-induced immunotoxic effects were due to an increase of redox status. In previous research, ERK1 and ERK2 have been demonstrated as ultimately phosphorylated products from MAPK families, and the activated ERKs translocate to the nucleus and transcription factors, changing gene expression to promote growth, differentiation, or mitosis.42 Also, the ERK signaling pathway is one of the major pathways of Tlymphocyte proliferation.43 However, there is little research available on the relationship with the ERK pathway and immunosuppression of AFB1. In our experiment, T cells were exposed to various concentrations of AFB1 (1−8 μg/mL), and expression of ERK1/2 was increased at 2, 4, and 8 μg/mL in a dose-dependent manner. Our current results strongly indicated that AFB1 exposure could activate the ERK1/2 signaling pathway in primary porcine splenocytes. Above all, hypotheses were formulated that the change of phosphorylation of ERK1/2 seems likely correlated with ROS generation in certain aspects. In the previous study, Keshari et al. demonstrated ROS resulted in activation of ERK in PMNs, which is an important component of the innate immune system.44 To examine oxidative stress as a function in the MAPK signaling pathway, NAC was added before AFB1 exposure. The finding that NAC, a nonspecific antioxidant, down-regulated the phosphorylation of the ERK1/2-MAPK pathway in primary porcine splenocytes (Figure 4 B) further certified this hypothesis. To further determine the specificity of the ERK1/2−MAPK pathway regulating AFB1-induced oxidative stress and immune toxicity, experiments were repeated using ERK-specific siRNA, which can specifically block ERK1/2 expression (Figure 6). O’Connell et al. made a key contribution to the inhibition of the MAPK pathway resulting in attenuation of the CsA-induced mesangial cell alterations caused by oxidative stress.45 Our results are consistent with the previous studies, which strongly indicate that ERK1/2-knockdown mitigates AFB1-induced cellular immunity toxicity and the ERK pathway maybe is a key mechanism of T-cell suppression induced by AFB1. It seems that AFB1 exposure produced oxidative stress, and then oxidative stress activated the ERK signal pathway and resulted in the suppression of T-cell proliferation. The mechanisms by which oxidative stress and ERK1/2 phosphorylation correlated with interaction are relatively unexplored but undoubtedly important areas for future investigation.

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AUTHOR INFORMATION

Corresponding Author

*(K.H.) Phone: +86-25-84395507. Fax: +86-25-84398669. Email: [email protected]. Funding

This work was funded by the National Natural Science Foundation of China (31272627, 31472253), the Research Fund for Doctoral Program of Higher Education in China (20110097110014 and 20120097130002), and the Priority Academic Program Development of Jiangsu Higher Education Institutions (Jiangsu, China). Notes

The authors declare no competing financial interest.

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REFERENCES

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DOI: 10.1021/acs.jafc.5b00433 J. Agric. Food Chem. XXXX, XXX, XXX−XXX