Hyperthermia Affects Immune and Oxidative Stress Indices of Immune Organs of Broilers by Changing the Expressions of ABCG2, SVCT-2 and MCU

Background: As global temperatures rise, heat stress has become one of the major environmental stressors in the poultry industry. The purpose of the study was to investigate the effects of heat stress on immune function and oxidative stress, and further reveal the possible mechanisms of oxidative stress induced by heat stress for thymus and spleen of broilers. Methods: At the age of 28 days, thirty broilers were randomly divided into the control group (25 ± 2°C; 24 h/day) and the heat stress group (36 ± 2°C; 8 h/day); the experience was lasted for 1 week. At the end of the experience, the broilers per group were respectively euthanized and collected some samples, then to be analyzed. Results: The results showed that the levels of heat shock proteins 70 (HSP70,P(cid:0) 0.01), corticosterone (CORT,P(cid:0) 0.01), the contents of malondialdehyde (MDA, P(cid:0) 0.05), interleukin-6 (IL-6, P(cid:0) 0.01) and tumor necrosis factor-alpha (TNF-α, P(cid:0) 0.01) in serum were signicantly higher in heat stress group than that in the control group; The activities of total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) and contents of glutathione (GSH) in heat stress group signicantly reduced (P(cid:0) 0.05) in serum. Compared with the control group, the birds subjected to heat stress reduced the weight (P(cid:0) 0.01) and the indices of thymus (P(cid:0) 0.01), the activities of T-AOC (P(cid:0) 0.01) and SOD (P (cid:0) 0.05) of spleen, and levels of IL-10 (P(cid:0) 0.05) and the GSH-PX (P(cid:0) 0.05) in thymus and spleen, and increased the IL-6 content of thymus (P(cid:0) 0.05), the MDA content (P(cid:0) 0.01), and the reactive oxygen species (ROS) levels (P(cid:0) 0.01) in thymus and spleen. Moreover, the expression of immunoglobulin G (IgG) gene in thymus and spleen of heat stressed broiler signicantly increased by reverse transcription-polymerase chain reaction (RT-PCR) and real time RT-PCR (qRT-PCR; P(cid:0) 0.05); However, the expression of immunoglobulin M (IgM) gene in spleen signicantly increased (P(cid:0) 0.05), and had no signicant difference (P(cid:0) 0.05) in thymus of heat-stressed broiler. Furthermore, the relative expression of ATP binding cassette subfamily G member 2 (ABCG2) in thymus and spleen (P(cid:0) 0.05), sodium dependent vitamin C transporter-2 (SVCT-2, P(cid:0) 0.01) and mitochondria calcium uniporter (MCU, P(cid:0) 0.01) mRNA in thymus of heat stressed broilers signicantly increased; and the expression of ABCG2 (P(cid:0) 0.05), SVCT-2 (P(cid:0) 0.01) and MCU (P(cid:0) 0.01) protein of thymus and spleen in the heat-stressed broiler increased signicantly compared with the control group. Conclusions: In summary, the study conrmed that heat stress caused oxidative stress to immune organs of broilers, further reduced immune function. Moreover, the potential mechanisms of heat stress-induced oxidative stress for thymus and spleen was further reveal in broilers. chain RT-PCR: reverse transcription-polymerase chain calcium uniporter; SVCT-2:sodium dependent vitamin transporter-2.


Introduction
The poultry industry is one of the largest among the industrie [1]. Heat stress has become one of the major environmental stressors in the poultry industry resulting in substantial economic loss due to global warming [2]. Heat stress causes increased mortality and reduced feed e ciency, body weight, feed intake and immunity [3]. Immune system is one of the main targets of heat stress-induced negative effects on the organism [4]. In chickens, the thymus and spleen are signi cant immunological organs and are respectively the major central lymphoid organ and the peripheral immune organ [5], and both are severally participate in the cellular and humoral immunity [6]. Heat stress could cause immune organ dysfunction and apoptosis [7], and reduce the weight of chicken spleen and thymus [8,9], then to inhibit their development by inducing the oxidative stress of organs in chickens [10][11][12]. The cause of heat-induced oxidative stress is mainly intracellular reactive oxygen species (ROS) production changes leading to the modi cation of the enzyme activity [13]. The hyperthermia could induce oxidative stress and is associated with augmented production of cellular ROS and oxidative damage to immune organ [8].
However, the mechanism of regulating ROS production in thymus and spleen of heat stressed broilers is not still fully understood.
ATP binding cassette (ABC) subfamily G member 2 (ABCG2) is a member of the ABC superfamily of proteins and plays an important role in drug resistance and maintaining cell homeostasis in the stress environment [14]. It has been found that ABCG2 is capable of protecting cells from ROS-mediated cell damage and death [15], and the downregulation of ABCG2 induces the overproduction of ROS to inhibit the production of antioxidants [16,17]. Moreover, vitamin C (Vc)in cells can eliminate free radicals to alleviate oxidative stress [18]. Sodium dependent Vc transporter (SVCT) is a kind of protein that exists on the membrane and has the function of transporting Vc [19]. SVCT-2 mainly participates in the absorption of Vc to protect tissue from oxidative damage [20]. Thus, we proposed that ROS levels of thymus and spleen may be dependent on the level of SVCT-2 expression in heat stressed broilers. Furthermore, calcium (Ca 2+ ) in cells is the second messenger of information transmission [21], and participates in oxidative stress and apoptosis [22]. The mitochondria calcium uniporter (MCU) is a selective channel that mediates the in ux of Ca 2+ on the inner membrane of mitochondria [23]. The change of MCU activity is closely related to ROS production and oxidative stress [24]. Therefore, we hypothesized that heat stress can change the expression of ABCG2, SVCT-2 and MCU, and further to regulate the production of ROS, then induce the oxidative stress of the thymus and spleen in broilers. Hence, in this study, we investigated that the effects of heat stress on immune and oxidative stress indexes of thymus and spleen, and the relationship between the expression of ABCG2, SVCT-2, MCU and the production of ROS was further analyzed. This will provide a therapeutic target for the later prevention of heat stress in broilers.

Broilers and Experimental Design
One-day-old Arbor Acres broilers were obtained from a commercial hatchery in Anhui, and kept in cages with wood shaving litter oor reared under routine commercial management practices. At the end of 28 days, thirty chickens were randomly divided into two groups (the control group and the heat stress group, 15 chickens /group), with 3 replicates per group, and 5 birds per replicate. Namely, the control group, kept at normal temperature conditions (25 ± 2°C; 42-66% RH for 1 week); while the heat stress group, exposed daily to high ambient temperature (36 ± 2°C; 33-38% RH, 8 h/day for 1 week, other time returned back to the conventional conditions) to mimic an environmental heat wave in an environmentally-controlled chamber. The heat exposure protocol was conducted for 1 week (from 28 to 35 days). The chickens were kept under constant light throughout the experiment, with feed and water being provided ad-libitum. Ingredients and chemical composition of the basal diet refer to the article by Wang et al., (2019) [25].

Sample collection and preservation
After 1 week of heat stress, the broilers for the control group and heat stress group were slaughtered, and then the blood samples were respectively collected from the jugular vein in tubes and immediately centrifuged at 3,000 rpm for 15 min to obtain serum, then stored at -20℃. The serum was further used to analyze the heat shock proteins 70 (HSP70), the corticosterone (CORT) and the indices of oxidative stress. The thymus and spleen were respectively collected and weighed, and then were divided into two parts. Part of the sample is quickly put into liquid nitrogen and then stored at -80°C for RNA isolation extract. The other part of the sample was immediately homogenized, and then to determine oxidative stress and immune indexes.

The levels of HSP70 and CORT determination in serum
The levels of HSP70 and CORT concentration in the serum were detected by using enzyme-linked immunoassay (ELISA) kit (Shanghai Fanke Industrial Co., Ltd., Shanghai, China); the procedure was followed as provided by the supplier.

Determination of oxidative stress and immune Indices in serum
After 1 week of heat stress, total antioxidant capacity (T-AOC), the activities of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD) and catalase (CAT), and the contents of the glutathione (GSH) and malondialdehyde (MDA) were determined using corresponding diagnostic kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the instructions of the manufacturer as previously described [26]. The levels of interleukin-6 (IL-6), interleukin-10 (IL-10) and tumor necrosis factor-alpha (TNF-α) in serum were detected by using a commercial ELISA kit (Nanjing Senberga Biological Technology Institute, Nanjing, China) according to the instructions of the manufacturer.

Measurement of oxidative stress and immune indices for thymus and spleen
The thymus and spleen were immediately collected and homogenized for 2 min in 9 mL of ice-cold saline solution, respectively, and then the homogenates were centrifuged at 3,000 rpm for 15 min at 4°C. The Relative quantitative real-time polymerase chain reaction analysis Reverse transcription polymerase chain reaction (RT-PCR) and qRT-PCR were performed by referring to previously described methods [25]. Brie y, the total RNA was respectively extracted from thymus and spleen of broilers using OMEGA Total RNA kit (SPECTRIS CO, Egham, Surrey, UK) according to the manufacturer's instructions, and then reverse transcribed into cDNA using Takara prime scripter kit (Takara Bio., Kusatsu, Shiga, Japan) following the instructions of the manufacturer. The cDNA was diluted and stored at -30℃ until gene analysis. The PCR products were examined using 1.5% agarose gel electrophoresis; β-actin was used as the internal standard in the study. The relative levels of IgG, IgM, ABCG2, SVCT-2 and MCU mRNA were respectively determined using CFX Connect™ Real-Time PCR Detection System (Bio-Rad, California, USA). The PCR procedure was 95℃ for 10 mins, and then followed by 40 ampli cation cycles of denaturation at 95℃ for 15 s, annealing at 60℃ for 1 min, and extension at 72℃ for 15 s. The melting curve shows a single peak for each PCR product. The relative levels of immunoglobulin G (IgG), immunoglobulin M (IgM), ABCG2, SVCT-2 and MCU mRNA were calculated according to the 2-△△CT method. According to the sequence of β -actin, IgG, IgM, ABCG2, SVCT-2, MCU and housekeeping gene β-actin of chicken in NCBI, the primers were designed by AlleleID6 (PREMIER Biosoft, Canada). The primers were synthesized by Shanghai Jierui biology Co., Ltd., as shown in Table 1. Table 1 Gene-special primers used in the RT-PCR and qRT-PCR.

Gene
Primer sequence(5′→3′) Product size (bp) Protein extraction and Western Blotting The thymus and spleen were respectively cut into small pieces in the homogenate tube, and then were homogenized for 2 min in 10 times tissue volume of lysate. After the homogenates were cracked on the ice for 30 mins, they were centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was collected as the total protein solution. The protein content of tissue homogenate was measured by a Coomassie brilliant blue staining (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The procedure was followed as the method provided by the supplier.
Tissue protein samples for the thymus and spleen were separated on a sodium dodecyl sulfate (SDS)polyacrylamide gel (PAGE) and transferred to polyvinylidene uoride (PVDF) membrane (Millipore, Billerica, MA) by electro blotting; After blocking with 5% skimmed milk for 1 h at room temperature, the proteins were labeled with primary antibodies overnight at 4℃; and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody at room temperature for 30 mins; Bound antibodies were detected with ECL Western blot detection reagent (GE Healthcare) and quanti ed with Fiji/Image J. Relative signal intensities were normalized to β-actin. All the primary antibodies and secondary antibodies were purchased from Santa Cruz (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Statistical analysis
All the data were analyzed by SPSS software (version 21; IBM, Chicago, USA), and expressed by means ± S.E.M. from 3 replicates. Statistical comparisons were performed using Independent-Samples T Test. A difference with value P< 0.05 was considered statistically signi cant.

Results
The levels of HSP70 and CORT, and oxidative stress and immune indices in serum after heat stress As shown in Table 2, after 1 week of heat stress, the levels of HSP70 and CORT in serum was signi cantly higher than that in the control group (P< 0.01); The birds had higher the contents of MDA in heat stress group compared with the control group (P< 0.05); The activities of T-AOC, GSH-Px and SOD and contents of GSH in heat stress group were signi cantly lower (P< 0.05) than those in the control. Moreover, the levels of IL-6 and TNF-α in heat stress group signi cantly increased in compared with the control group (P< 0.01); while the CAT activity and IL-10 level had no signi cant difference between the heat stress and the control groups (P> 0.05). Changes of development and immune indices of thymus and spleen after heat stress As shown in Table 3, the weight and the indices of thymus in the heat stress group were signi cantly lower than those in the control group (P< 0.01). There was no signi cant difference in the weight and indices of spleen between the heat stress and the control groups (P> 0.05). As shown in Table 4, compared with the control group, the IL-6 contents of thymus in the heat stress group was signi cantly increased (P< 0.05), had no signi cant change in spleen (P> 0.05); The IL-10 levels of thymus and spleen in the heat stress group was both signi cantly lower (P< 0.05); The contents of TNF-α was no signi cant difference in thymus and spleen (P> 0.05).  Indices of oxidative stress in thymus and spleen of broilers under heat stress As shown in Table 4, the MDA content and the ROS levels (measured as H 2 O 2 ) of thymus and spleen in heat stress group was signi cantly higher than that in the control (P< 0.01); Compared with the control birds, the activities of T-AOC (P< 0.01) and SOD (P< 0.05) for spleen in heat stress group were signi cantly lower, but they had both no signi cant change in thymus (P> 0.05); Moreover, the GSH-PX content of thymus (P< 0.05) and spleen (P< 0.01) in heat stress group was signi cantly lower than that in control group. The results showed that heat stress could cause the increase of lipid peroxides and ROS, the decrease of antioxidant enzymes in thymus and spleen of broilers.

Expression of IgG and IgM mRNA in thymus and spleen of broilers
To understand whether heat stress can induce immune response in the thymus and spleen of broilers or not, the expression of IgG and IgM genes was detected by RT-PCR and RT-qPCR. As shown in Fig. 1A, the expression of IgG and IgM genes in heat stress group signi cantly increased compared to the control group in thymus and spleen by RT-PCR; The relative expression of IgG and IgM mRNA in heat stress group signi cantly increased compared to the control group in spleen (P< 0.05; Fig. 1B); In the thymus, the expression levels of IgG mRNA in heat stress group was signi cantly higher than that in the control group (P< 0.05); However, the expression level of IgM mRNA had no signi cant difference (P> 0.05; Fig. 1B).

Expression of ABCG2, SVCT-2 and MCU genes in thymus and spleen of broilers under heat stress
To further explore the reasons for the change of ROS levels of thymus and spleen caused by heat stress, the gene expression of several transporters involved in ROS production was examined by RT-PCR and RT-qPCR. As shown in Fig. 2A, the birds exposed to heat stress had higher expression of ABCG2, SVCT-2 and MCU mRNA compared with the control birds in thymus and spleen by RT-PCR analysis. The relative expression of ABCG2 mRNA in heat stress group increased in thymus and spleen (P< 0.05) (Fig. 2B). Furthermore, the relative expression of SVCT-2 and MCU mRNA in thymus of heat stressed broilers signi cantly increased compared with the control birds (Fig. 2C,D) (P< 0.01), but it had no signi cantly change in spleen (P> 0.05).

The expression of ABCG2, SVCT-2 and MCU proteins in thymus and spleen of broilers under heat stress by Western Blot
To clarify the reason of ROS production changes in thymus and spleen after heat stress, the expression of several transporters involved in ROS production was examined by Western Blot. As shown in Fig. 3A, the expression of ABCG2, SVCT-2 and MCU proteins were all dramatically increased in thymus and spleen after exposure to heat stress. The expression of ABCG2 (P< 0.05; Fig. 3B), SVCT-2 (P< 0.01; Fig. 3C) and MCU (P< 0.01; Fig. 3D) protein of broiler thymus and spleen in the heat stress group increased signi cantly compared with the control group. The results suggest that heat stress may mediate oxidative stress in the thymus and spleen of broilers by regulating the expression of ABCG2, SVCT-2 and MCU protein.

Discussion
Environmental temperature plays an important role in poultry industry along with rising of the global temperatures. When the environmental temperature exceeds the upper limit of comfort zone of broilers by 27℃, the imbalance between heat dissipation and heat production of the body will lead to heat stress [27]. Hence, the broiler were exposed at 36℃ for 8 hours per day and lasted for 1 week to mimic a heat wave in the study. The broiler in heat stress group showed shortness of breath, wing spreading, lethargy and increased water consumption at this study. The results are consistent with those reported by Jastrebski et al. (2017) [28]. Previous studies have shown that HSP70 is one of the most conservative proteins in evolution and very stable at heat stress, and can be used as stress indicator protein [29]. Heat stress can signi cantly increase the expression of HSP70 in broiler cardiomyocytes [30]; moreover, the content of serum CORT is the most important index of heat stress in poultry [31]. Hence, the content of HSP70 and CORT in the serum was used to judge that the broilers had heat stress, and they in heat stress group were signi cantly higher than that of the control group in the study. In the course of our experiment, the broilers showed a typical heat stress response in the early stages of thermal exposure.
Heat stress is linked to compromised productivity through the change of biochemistry and immunity in blood [32]. It's important to note that immune system is one of the main targets of heat stress-induced negative effects in the organism [4]. The spleen is the biggest peripheral immune organ, and thymus is central lymphoid organs in the immune system of poultry [33]. Hence, we selected spleen and thymus as the target tissues to analyze the mechanism of change of ROS production for heat-stressed birds in the study. Previous studies have con rmed that heat stress affected the absolute weight and organ index of immune organs to a certain extent [11]. In the current study, the absolute weight and index of thymus and spleen all decreased after heat stress; this indicate in the immune system is inhibited to some extent, which is consistent with previous research results [6,34]. The results showed that heat stress could inhibit the development of thymus and spleen and cause damage to the immune organs.
Cytokines are important information molecules of the immune system in the immune response process of the body [35]. Recent studies have shown that IL-6 and TNF-α, as proin ammatory factors, play important roles in both immune responses and in ammationin, which can activate immune response and in ammation [36,37]. IL-10 is an important anti-in ammatory cytokine and can inhibit production of in ammatory cytokines by a variety of in ammatory cells [38]. Studies have shown that IL-10 can inhibit the activation, synthesis and release of proin ammatory factors such as IL-6 and TNF-α through various mechanisms, so as to achieve the purpose of inhibiting the occurrence of in ammation [39]. Our present study showed that the levels of IL-6 and TNF-α in serum for heat stressed broilers increased, but the IL-10 level had no signi cant change. Moreover, heat stress can increase the level of IL-6 and TNF-α, and decrease the level of IL-10 in thymus and spleen of broilers. The results con rmed that heat stress can increase the levels of in ammatory cytokines and decrease the levels of anti-in ammatory cytokines in serum,the thymus, and spleen of broilers.
Besides these cytokines, immunoglobulin also plays an important role in immune regulation. Existing research has demonstrated that IgM and IgG are respectively the immunoglobulin in poultry, which play a leading role in anti-infection [40]. The relative expression of IgM and IgG mRNA in the spleen and thymus of heat stressed broilers increased signi cantly in the study. These results agree with previous studies reported by Honda et al. (2015) [40]. This is an indication that heat stress caused infection to the immune organs; further enhanced the immune response of spleen in broilers. Hence, heat stress can lead to in ammatory response and immunoglobulin expression increase in thymus and spleen.
Heat stress affects mainly poultry performance by inducing the oxidative stress [41]. Previous studies had con rmed that MDA content can re ect the degree of oxidative damage [42]. In this study, we found that the MDA content of thymus and spleen increased signi cantly under heat stress, which indicated that heat stress caused oxidative damage of thymus and spleen in broiler. Existing research has demonstrated that hyperthermia causes oxidative stress damage to the cell by generating augmented ROS [8], while SOD and GSH-PX can remove ROS in the body [43]. In this experiment, the contents of SOD, GSH-PX and T-AOC were decreased, while the ROS in thymus and spleen of broiler increased signi cantly after heat stress, which indicated that heat stress could reduce the antioxidant capacity and lead to the increase of ROS level in thymus and spleen, and causes oxidative injury in the cell of the thymus and spleen.
To explore the mechanism of change of ROS levels in the thymus and spleen of heat-stressed birds, the expression of several transporters in the thymus and spleen associated with ROS production was detected. ABCG2 often serves as an important marker for cells in oxidative stress [44] and is capable of protecting cells from ROS-mediated cell damage [16]. It has been found that ABCG2 can reduce the ROS levels to alleviate the oxidative stress of cells [15] and improve the content and activity of SOD, the content of glutathione and the activity of glutathione reductase in cells [45]. Our study found that the expression of ABCG2 mRNA and proteins in thymus and spleen of heat stressed broiler was signi cantly increased. The data showed that ABCG2 expression manifested as a compensatory increase to protect the thymus and spleen from heat-induced oxidative stress by reducing intracellular ROS or exuding other harmful substances under heat stress. However, more in-depth research is needed in the future to articulate the anti-injury mechanism of ABCG2 in heat stress. Moreover, SVCT-2 widely exists in various tissues and organs, which mainly participates in the absorption of vitamin C in the active metabolism tissues in the body, so as to protect these tissues from oxidative damage [20]. Previous studies have indicated that supplementation with vitamin C could mitigate heat-related damage and enhance heat resistance in animals [46]. It has been shown that SVCT-2 gene knockout can reduce the content of Vc in cells and tissues, increase cell oxidative damage and death, and nally lead to embryo death [47]. Our studies found that the expression of SVCT-2 mRNA in thymus increased, while it is no signi cant different in the spleen of the heat-stressed broiler. However, the expression of SVCT-2 proteins in thymus and spleen of heat stressed broiler was signi cantly increased. The result indicated that SVCT-2 proteins are stimulated to possibly transfer more Vc and to compensatory to protect against cellular damage of thymus and spleen from the heat-generated stress; however, further research is needed to elucidate the exact mechanism.
Ca 2+ is a second messenger that mediates cell apoptosis and oxidative stress. A previous study showed that Ca 2+ dysregulation can give rise to neurodegenerative diseases through oxygenated stress damage [48]. The MCU has the function of mediating Ca 2+ in ux and plays an important role in the regulation of Ca 2+ concentration in cells and mitochondria [23]. Since MCU is associated with Ca 2+ concentration, while mitochondrial Ca 2+ overload induces a large number of ROS production in cells [49]. Our study showed that the expression of MCU in thymus and spleen of heat stressed broiler was signi cantly increased compared with the control group. This indicated that under the condition of heat stress, Ca 2+ overload of mitochondria in thymus and spleen cells of broiler maybe affect ATP synthesis, further stimulated ROS production, and nally led to apoptosis, thus affecting immune function.

Conclusions
In conclusion, chronic heat stress can cause oxidative stress in thymus and spleen of broilers. After heat stress, the body will over-express ABCG2 and SVCT-2 to clear ROS and protect spleen from oxidative damage to a certain extent. However, the decreased expression in thymus may explain that heat stress is more harmful to broiler thymus. While the expression of MCU in the thymus and spleen of broilers increased signi cantly. The opening of MCU will lead to mitochondrial calcium overload, stimulate ROS production, and lead to apoptosis or necrosis of thymus and spleen cells, which will affect the immune function of broilers. Availability of data and materials The datasets analyzed during the study are available from the corresponding authors upon reasonable request.
Ethics approval and consent to participate All experimental protocols were approved by the Animal Care and Use Committee of Anhui Agricultural University. The methods were carried out in accordance with the approved guidelines.

Consent for publication
Not applicable.
The expression of IgG and IgM mRNA in thymus and spleen by RT-PCR and RT-qPCR analysis. A. Expression of IgG and IgM genes by RT-PCR analysis; B. Relative mRNA expression levels of IgG in thymus and spleen; C. Relative mRNA expression levels of IgM in thymus and spleen. CG, the control group. HS, heat stress group. Note: Compared with the control group, *P < 0.05, ** P < 0.01. Error bars, SEM.  Compared with the control group, *P < 0.05, ** P < 0.01. Error bars, SEM.