Introduction

Lead (Pb), a highly toxic metal, is commonly found in both biotic and abiotic components in an ecosystem. Currently, possible sources of Pb in the poultry industry include lead solder in food cans and lead-arsenate pesticides, among others [1]. Pb exposure may cause various physiologic changes, affecting hematopoietic, reproductive, vascular, and physiological functions. Additionally, Pb may cause carcinogenesis, mutagenesis, and teratogenesis in experimental animals [2]. In humans, Pb affected different systems and caused neurological symptoms such as headache, lethargy, peripheral neuropathy, severe convulsions, encephalopathy, and even coma [3]. In poultry, broiler chickens are vulnerable to Pb intoxication. Even 1.0 ppm Pb in the diet could cause significant growth suppression and consistent declines in blood D-aminolevulinic acid dehydratase and in an erythrocyte enzyme sensitive to Pb in chickens [1]. Lead can cause adverse effects on the immune system. Pb exacerbates systemic lupus erythematosis (SLE) symptoms in lupus-prone NZM mice [4]. Lead could also affect cells involved in cell-mediated immunity, such as neutrophil leukocytes [5]. Specifically, Pb inhibited in vitro neutrophil chemotaxis, phagocytosis, and superoxide formation and impaired cell chemotaxis [6]. Cytokines are regulatory proteins that play a primary role in modulating innate and adaptive immune responses, including lymphocyte activation, proliferation, differentiation, and apoptosis. They can be classified as interleukins, such as interleukin-4 (IL-4) and interleukin-1 (IL-1); interferons, such as interferon gamma (IFN-γ); colony-stimulating factors; tumor necrosis factors; tumor growth factors; and chemokines. Cytokines might be affected by heavy metals at levels of exposure that do not affect other constituents of the immune system [7]. A number of cytokines govern neutrophil functions in humans [8]. Heat shock proteins (HSPs), a kind of stress protein that exists in all kinds of cells in organisms, have the ability to modulate cellular anti-stress responses and play a key role in protecting organisms from metal stress [9]. HSP70 is one of the biomarkers of oxidative stress induced by heavy metals. A study found that the expression of HSP27 and HSP70 was increased in chromium-treated mice livers [10].

Selenium (Se) is an essential trace element for organisms. Selenoproteins have a crucial role in the maintenance of cellular redox homeostasis in nearly all tissues and are also involved in thyroid hormone metabolism, inflammation, and immunity [11]. Se is a well-established antioxidant and can prevent or decrease the harmful effects of heavy metal on the antioxidant system in different tissues [12]. The protective effect of Se against the toxicity of different heavy metals in biological systems has been studied [13]. Se can counteract the toxicity of various elements, especially Hg2+ and MeHg [14]. Se has been shown to antagonize lead-induced neurotoxicity through the regulation of uptake and excretion of Pb and to prevent damage induced by oxygen free radicals [15]. Supplementation with a suitable dose of Se could alleviate the effects of Pb toxicity in the brain and blood of rats [16]. Moreover, Se is known to coexist with Hg in fish and sea mammals [17] and might play a role in antagonizing MeHg toxicity. Li et al. have already demonstrated the protective effects of Se against sub-chronic exposure to dietary Cd, which could cause hepatotoxicity, oxidative stress, and apoptosis in chicken liver [18]. Ebselen, a seleno-organic compound, could inhibit Mn-induced ROS generation and was efficacious in reducing Mn-induced neuroinflammation, oxidative stress, and locomotor activity impairments [19]. Generally, studies on the antagonism of Se toward toxicity induced by Pb in chickens are mainly focused on its antioxidant function. However, its effects on cytokines and HSP pathways have rarely been reported. Thus, we designed the current experiment to investigate the effects of Pb on the cytokines and HSPs in neutrophils of chicken peripheral blood and to examine the antagonistic effects of Se against lead.

Materials and Methods

Animal Model and Experimental Design

A total of 180 local 1-day-old male Hy-Line brown chickens were randomly divided into four groups (n = 45). Each group was randomly allocated to three pens (15 chickens per pen). During the entire experimental period, the chickens were given free access to food and water. In the present study, we fed chickens a basal diet or a diet supplemented with Pb, Se, or Pb+Se. The control group (Con group) was maintained on a basal diet containing 0.49 mg kg−1 Se. The Se-adequate group (+Se group) was maintained on a Se-supplemented basal diet containing 1.00 mg kg−1Se and conventional drinking water, while the Pb-supplemented group (+Pb group) was maintained on a basal diet with 350 mg kg−1 lead added to the drinking water. The Se and Pb compound group (Se+Pb group) was maintained on a Se-supplemented basal diet containing 1.00 mg kg−1 Se with 350 mg kg−1 Pb added to the drinking water. Se was added as sodium selenite, and Pb was added as lead acetate. All procedures used in this experiment were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University. Chickens were euthanized at 90 days. The whole blood was quickly removed, and the neutrophils were separated immediately and stored at − 80 °C until subsequent experiments.

In this study, the neutrophils were collected at five sampling events, and five chickens per group were used in the official test (n = 5), which was repeated in triplicate. The neutrophils were separated using chicken peripheral blood neutrophil isolation kits according to the manufacturer’s instructions (TBD, China).

Primer Design

The primer sequences of cytokines IL-1β, IL-1R, IL-2, IL-4, IL-8, IL-10, IL-12, IFN-γ, and TGF-β4 and of HSPs HSP27, HSP40, HSP60, HSP70, and HSP90 published in GenBank (Table 1) were synthesized by the Invitrogen Biotechnology Co. Ltd. in Shanghai, China. GAPDH was used as an internal reference gene.

Table 1 Gene-special primers used for qPCR
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Total RNA Isolation and Reverse Transcription

The total RNA was isolated from neutrophil samples using Trizol (Invitrogen, China) according to the manufacturer’s instructions. The dried RNA pellets were resuspended in 40 μl of diethylpyrocarbonate-treated water. The concentration and purity of the total RNA was determined spectrophotometrically at 260/280 nm (Gene Quant 1300/100, USA). The total RNA was immediately used to synthesize first-strand cDNA using the AccuPower® RocketScript™RT PreMix (BIONEER) according to the manufacturer’s instructions. Reverse transcription (RT) reactions (60 μL) consisted of the following: 6 μL of total RNA, 1.5 μL of RNase inhibitor, 3 μL of dNTP mixture, 3 μL of 20× OligodT(25), 3 μL of Golden MLV Reverse Transcriptase, 6 μL of 10× RT buffer, and 37.5 μL RNase Free H2O. The RT procedure was carried out for 15 min at 30 °C followed by 50 min at 55 °C and 10 min at 85 °C. The RT products (cDNA) were diluted five times with sterile water and stored at − 80 °C until use.

Quantitative Real-Time PCR (qPCR)

The qPCR was performed with Light Cycler® 96 (Roche Life Science). Reactions were performed in a 10-μL reaction mixture containing 5 μL of 2× SYBR Green PCR Master Mix (Roche, Switzerland), 1 μL of diluted cDNA, 0.3 μL of each primer (10 μM), and 3.4 μL of PCR-grade water. The qPCR procedure for HSPs, inflammatory genes, and β-actin consisted of heating the reaction mixture to 52 °C for 2 min and 95 °C for 10 min followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min, 95 °C for 15 s, and 60 °C for 20 s. The melting curve analysis showed only one peak for each PCR product.

Western Blot Analysis

Protein extracts were subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions on 12% gels. Separated proteins were then transferred to nitrocellulose membranes using a tank transfer for 60 min at 200 mA in Tris-glycine buffer containing 20% methanol. Membranes were blocked with 5% skim milk for 16–24 h and incubated overnight with the diluted primary chicken antibodies HSP60 and HSP70(1:1000, production of polyclonal antibody by our lab) followed by a horseradish peroxidase (HRP)-conjugated secondary antibody against rabbit IgG (1:2000, Santa Cruz, CA, USA). To verify equal loading of samples, the membrane was incubated with monoclonal β-actin antibody (1:1000, Santa Cruz, CA, USA) followed by a HRP-conjugated goat anti-mouse IgG (1:2000). The signal was detected using X-ray films (Trans Gen Biotech Co., China). The optical density (OD) of each band was determined using the Image VCD gel imaging system, and iNOS expression was detected as the OD ratio of HSP60 and HSP70 to β-actin.

Statistical Analysis

Statistical analysis of all data was performed using SPSS for Windows (version 19, SPSS Inc., USA). The data was analyzed using a one-way analysis of variance, and measurements were considered significantly different when P < 0.05. All data were checked for normal distribution and equal variance. Differences between means were assessed using Tukey’s honest significant difference test for post hoc multiple comparisons. The data were expressed as the mean ± standard deviation. All experiments were performed a minimum of three times. In addition, principal component analysis (PCA) was used to define the most important parameters, which could be used as key factors for individual variations using the Statistics 6.0 program (version 19, SPSS Inc., Chicago, IL, USA).

Results

Effects of Se, Pb, and Se+Pb on the mRNA Levels of Cytokines in Chicken Neutrophils

The mRNA levels of cytokines (IL-1β, IL-1R, IL-2, IL-4, IL-8, IL-10, IL-12, IFN-γ, and TGF-β4) in chicken neutrophils were measured by RT-PCR (Fig. 1). Compared with the control group, the mRNA expression levels of IL-1β, IL-1R, IL-4, IL-8, IL-10, IL-12, and TGF-β4 were significantly increased (P < 0.05) in the +Pb group, and significant decreases were also observed in the mRNA levels of IL-2 and IFN-γ of the +Pb group (P < 0.05). However, compared with the +Pb group, treatment with Se significantly reduced the mRNA expression of IL-1β, IL-1R, IL-4, IL-8, IL-10, IL-12, and TGF-β4 and increased the mRNA expression of IL-2 and IFN-γ in the Se+Pb group (P < 0.05).

Fig. 1
figure 1

Effects of dietary Se and Pb on the mRNA levels of cytokines in chicken neutrophils. The relative mRNA expression levels compared to the control group were used as the reference values. Each value is represented as the mean ± SD (n = 5). Bars not sharing a common letter indicate that there were significant differences between the two groups (P < 0.05)

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Effects of Se and Pb on the mRNA Levels of HSPs in Chicken Neutrophils

The mRNA levels of HSPs in chicken neutrophils were detected by RT-PCR (Fig. 2). Compared with the control groups, the mRNA levels of HSP27, HSP40, HSP60, HSP70, and HSP90 were significantly higher (P < 0.05) in the +Pb group. However, compared to the +Pb group, there was a significant decrease in the mRNA levels of HSPs in the Se+Pb group (P < 0.05), though the values were not restored to the levels of the control group.

Fig. 2
figure 2

Effects of Se and Pb on the mRNA level of HSP genes in chicken neutrophils. The relative mRNA expression levels compared to the control group were used as the reference values. Each value is represented as the mean ± SD (n = 5). Bars not sharing a common letter indicate that there were significant differences between the two groups (P < 0.05)

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Effects of Se and Pb on the Protein Levels of HSPs in Chicken Neutrophils

The protein levels of HSP60 and HSP70 in chicken neutrophils were examined using Western blots (Fig. 3). The results showed that, compared with the control group, Pb treatment significantly increased the protein expression of HSP60 and HSP70 (P < 0.05). However, compared with the +Pb group, Se supplementation notably reduced these protein levels in the Se+Pb group, though the values were not restored to the levels of the control group (P > 0.05).

Fig. 3
figure 3

Effects of Se and Pb on the protein level of HSP genes in chicken neutrophils. The relative protein expression levels compared to the control group were used as the reference values. Each value is represented the mean ± SD (n = 5). Bars not sharing a common letter indicate that there were significant differences between the two groups (P < 0.05)

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Principal Component Analysis

Using PCA, nine cytokines (IL-1β, IL-1R, IL-2, IL-4, IL-8, IL-10, IL-12, IFN-γ, and TGF-β4) and five heat shock proteins (HSP27, HSP40, HSP60, HSP70, and HSP90) from four treatment groups of chicken neutrophils were analyzed. A 3D plot of the PCA loadings is presented in Fig. 4 that clearly indicates correspondence to the first, second, and third principal components (86.153, 6.546, and 4.918%, respectively) in chicken neutrophils.

Fig. 4
figure 4

Principal component analysis of IL-1β, IL-1R, IL-2, IL-4, IL-8, IL-10, IL-12, IFN-γ, TGF-β4, HSP27, HSP40, HSP60, HSP70, and HSP90

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As shown in Table 2, all the cytokines and heat shock proteins had closer relationships with the first principal components. The results revealed that HSP40, HSP60, HSP70, and HSP90 had a close positive correlation with each other in chicken neutrophils. Furthermore, of all the parameters that were measured in the present study, only IL-2 and IFN-γ had negative correlations with another parameter, while IL-1β, IL-1R, IL-4, IL-8, IL-10, IL-12β, TGF-β4, HSP27, HSP40, HSP60, HSP70, and HSP90 had close positive correlations with each other in chicken neutrophils.

Table 2 Correlation matrix
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Discussion

Pb is a common industrial poison and an environmental pollutant. A great deal of experimental data has suggested that acute and chronic exposure to inorganic lead may result in the impairment of immune functions in animals. For example, Pb triggered humoral and cellular immune response depressions in rodents [20]. Neutrophil, a type of phagocyte, is an essential part of the innate immune system. A previous study has demonstrated that lead exposure could inhibit neutrophil function [21]. Cytokines are effectors in the immune system that mediate activating and regulatory functions, which are important for immune responses [22]. Interleukins are a group of cytokines that are critical to the functioning of the immune system. Evidence suggests that the abnormal expression of interleukins could be induced by heavy metals and might impair immune function. For instance, IL-1R is involved in multiple immunological and inflammatory processes [23]. IL-1β is considered as an acute phase reactant; it is associated with fever, inflammation, tissue destruction, and occasionally even shock and death as an endogenous pyrogen [24]. Xu reported that cadmium caused a significant increase in IL-1β in chicken spleen lymphocytes [25]. IL-2 is related to the proliferation of B cells and enhances cytokine production of natural killers and neutrophils. It has been reported that suppressed IL-2 levels were associated with a general decline in immune function and immune regulation [26]. Liu X et al. has reported that the mRNA expression of IL-2 was also suppressed by manganese in Hy-Line brown cocks [27]. Total IL-4 is a known marker for humoral immune responses [28]. IL-8 is a member of the CXC chemokine subfamily. Neutrophils are major specific targets for IL-8 action, and the pathophysiological actions of IL-8 depend on the activation of neutrophils [29]. Pb toxicity affects the body systems by inducing changes in IL-8 [30]. Lin et al. proved that Pb2+ induced IL-8 gene expression by extracellular signal-regulated kinases [31]. IL-10 inhibits monocyte/macrophage and neutrophil generation and promotes a shift from the Th1 phenotype to the Th2 phenotype in T lymphocytes [32]. Villanueva et al. confirmed that Pb ions could inhibit the activities of Th1 cells but could increase the activity of Th2 cells [33]. IL-4 and IL-10 are secreted by Th2 cells, and Pb ions increase the expression of IL-4 and IL-10. IL-12 is a primary cytokine secreted by antigen-presenting cells. TGF-β expression inhibits IFN-γ generation in human vascular cells [34]. TGF-β4 is produced by Treg cells converted from CD4+Th17 cells [35]. TGF-β mRNA expression was significantly increased in the kidney of Chinook salmon exposed to CPF. Parenthetically, it has been observed that TGF-β4 mRNA significantly increases in the duodenum of chickens infected with Eimeria acervulina [36]. Finally, IFN-γ activates neutrophils, NK cells, and vascular endothelial cells. The role of IFN-γ in mediating the cellular immune response has been identified [37]. Kaminska et al. have proven that leukocyte production of IFN-γ in vitro could be significantly reduced by different concentrations of lead in cows [38]. It has been documented that lead could inhibit the Th type 1 (Th1) response, which is characterized by IL-2 and IFN-γ secretion and the promotion of cellular cytotoxic immunity [7]. In the present study, we also detected expression of other cytokines. These results showed that Pb treatment significantly increased the expression of IL-1β, IL-1R, IL-4, IL-8, IL-10, IL-12, and TGF-β4 and decreased that of IL-2 and IFN-γ, which is consistent with the previous studies. It suggested that Pb treatment could induce neutrophil injury and thereby impair immune function in chickens.

HSPs are a family of proteins that are produced by cells in response to exposure to stressful conditions, including heavy metal exposures [39]. The level of HSP mRNA (HSP27, HSP40, HSP60, HSP70, and HSP90) was significantly increased in the immune organs when chickens were exposed to arsenic trioxide [40]. Pb poisoning also induced mRNA expression of HSPs (HSP27, HSP40, HSP60, HSP70, and HSP90) in chicken peripheral blood lymphocytes [41]. Pb administration in Wistar rats also increased expression of HSP70 in the brain [42]. The increased expression of HSP90 has been reported to be a major mechanism for Pb resistance [43]. Consistent with the previous studies, our results showed that the mRNA for the five HSPs (HSP27, HSP40, HSP60, HSP70, and HSP90) was upregulated. It elucidated that Pb could also have an adverse influence on neutrophils by increasing the mRNA levels of HSPs to further impair the immune system of chickens.

Se plays an important biological role in humans and animals, including in immune function enhancement, by increasing the number of cytokines, such as interferon. For example, Seda et al. reported that Se at 0.625 mg/kg/day decreased IL-1β levels during the treatment of chemia-reperfusion-induced brain injury in rats [44]. The results agreed with Montgomery et al., who suggested that Se might enhance the immunity of young foals through increasing the expression of IL-2 [45]. Additionally, Se could also antagonize the toxic effects of heavy metals [46, 47]. Se could also ameliorate cadmium (Cd)-induced brain damage in chickens [48] and exhibited protective effects on the chronic poisoning and decreased HSP70 mRNA levels induced by arsenic in rat livers [49]. Se exhibited significant antagonistic roles against Pb-induced increases of HSP expression in chicken liver [50].

In the current study, with Se supplementation, the increased levels of IL-(1β, 1R, 4, 8, 10, and 12) and TGF-β4 mRNA and the decreased levels of IL-2 and IFN-γ mRNA induced by Pb were returned nearly to the levels seen in the control group. The increased levels of HSPs induced by Pb were similarly mitigated by Se supplementation. Our results were in agreement with previous studies. They illustrated that Se could antagonize Pb toxicity to recover the immune system function by affecting the expression of the cytokines IL-(1β, 1R, 2, 4, 8, 10, and 12), IFN-γ, and TGF-β4 and of HSP (27, 40, 60, 70, and 90).

As shown in the results of the PCA, there is a correlation between nine cytokines and the five HSPs. IL-2 and IFN-γ are negatively correlated with other parameters, but there is a positive correlation between IL-(1β, 1R, 4, 8, 10, and 12), TGF-β4, and HSP (27, 40, 60, 70, and 90). However, the correlations between cytokines and heat shock proteins were different. For example, IL-1β is highly related to HSP40, HSP60, and HSP70, but the correlation between IL-2 and HSP90 is small. Thus, we suggest that Pb poisoning not only affects the expression of cytokines and HSPs but also affects the expression in the two genes.

In conclusion, Pb could increase the expression of IL-(1β, 1R, 4, 8, 10, and 12) and TGF-β4, decrease that of IL-2 and IFN-γ, and increase that of HSP (27, 40, 60, 70, and 90) to induce neutrophil injury and thereby impair the immune system of chickens. However, Se could antagonize Pb toxicity in neutrophils in chickens.