Ir J Med Sci DOI 10.1007/s11845-013-0928-8

ORIGINAL ARTICLE

Arsenic trioxide alleviates airway hyperresponsiveness and promotes apoptosis of CD4+ T lymphocytes: evidence for involvement of the ER stress–CHOP pathway K. Li • L. Zhang • X. Xiang • S. Gong • L. Ma • L. Xu • G. Wang • Y. Liu • X. Ji S. Liu • P. Chen • H. Zeng • J. Li

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Received: 30 March 2012 / Accepted: 15 February 2013 Ó Royal Academy of Medicine in Ireland 2013

Abstract Background Asthma is a chronic inflammatory disorder of the airway. Arsenic trioxide (ATO) is an ancient Chinese medicine, which is used to treat psoriasis, asthma, and acute promyelocytic leukemia. Aim We wanted to research the effect of arsenic trioxide on asthma. Methods Using a murine model of asthma, the airway hyperresponsiveness was conducted by the Buxco pulmonary function apparatus. Total cell counts of BALF were counted with a counting chamber. Histopathological analysis of lung tissues was conducted by hematoxylin– eosin or periodic acid-schiff stain. CD4?T cells were purified from the spleen by positive selection, using immunomagnetic beads. Apoptosis measurements were done with Annexin-V/PI staining. Western blot analysis and real time-PCR were performed to assess the expression of C/EBP-homologous protein (CHOP) and glucose-regulated protein 78 (GRP78), respectively. RNA interference was conducted to inhibit the expression of CHOP. Results We found that arsenic trioxide treatment alleviated airway hyperresponsiveness and reduced inflammation of the lung in asthmatic mice. Furthermore, arsenic trioxide treatment promoted apoptosis of CD4?T cells in vivo and in vitro. When CD4?T cells were cultured with arsenic trioxide for 5 h at a concentration of 5 lM, the expression of GRP78 and CHOP was increased. Treatment of CD4?T

K. Li  L. Zhang  X. Xiang  S. Gong  L. Ma  L. Xu  G. Wang  Y. Liu  X. Ji  S. Liu  P. Chen  H. Zeng  J. Li (&) Department of Respiratory Medicine, The Second Xiangya Hospital of Central South University, Changsha, Hunan 410011, China e-mail: [email protected]

cells with CHOP siRNA, provided partial resistance to arsenic trioxide-induced apoptosis of CD4?T cells Conclusions These data demonstrated that arsenic trioxide can reduce the severity of asthma attacks and induce the apoptosis of CD4? T cell which the ER stress–CHOP pathway involved. Keywords Arsenic trioxide  Asthma  CD4? T cells  Apoptosis  CHOP

Introduction Asthma is a chronic inflammatory disorder of the airway characterized by airway hyperresponsiveness (AHR), inflammation, and airway remodeling [1]. Activated CD4? T cells play a crucial role in the initiation, progression, and persistence of asthma through promotion of airway inflammation and induction of airway hyperresponsiveness. Persistent airway inflammation is caused by decreased apoptosis of T cells and eosinophils. In asthma, T cells exhibit decelerated spontaneous apoptosis [2]. Prolonged survival of inflammatory cells may contribute to the respiratory symptoms of asthma. Arsenic trioxide (ATO) has long been considered a poison. Adverse health effects caused by arsenic trioxide include neurotoxicity, liver injury, and increased risk of cancer [3]. In traditional Chinese medicine, arsenic trioxide has been used to treat psoriasis, rheumatosis, and asthma. In Western medicine, arsenic trioxide was introduced as a treatment for acute promyelocytic leukemia (APL) and showed prodigious effectiveness. Previous studies also found that arsenic trioxide treatment could promote apoptosis of pulmonary eosinophils [4], and improve pulmonary function in a mouse model of asthma [5]. But the ability of

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arsenic trioxide to promote the apoptosis of CD4? T cells is unknown. It is now well accepted that cell apoptosis can result from activation of three major pathways: the extrinsic, intrinsic, and the most recently identified endoplasmic reticulum (ER) stress-mediated pathway. A number of biochemical and physiological stimuli, such as arsenic trioxide, can induce ER stress, at which point cells initiate self-protective mechanisms via activation of various stressresponse signaling pathways, termed the unfolded protein response (UPR). However, excessively strong or long-term ER stress can lead to cell apoptosis [6]. Glucose-regulated protein 78 (GRP78) and growth arrest DNA damage-inducible gene 153 (GADD153)/CPEBP homologous protein (CHOP) are two key markers of ER stress. GRP78 is involved in the UPR and protective mechanism during ER stress; while, CHOP is involved in the injury mechanism induced by excessive ER stress [7]. Thus, upregulation of the aforementioned markers is an indication of ER stress. Many reports have focused on apoptosis induction by arsenic trioxide; however, few studies have investigated the relationship between arsenic trioxide and ER stress. In the present study, we found that in a murine model of asthma, administration of arsenic trioxide can alleviate airway hyperresponsiveness. This effect of arsenic trioxide may relate to its ability to induce the apoptosis of CD4? T cells. Our study also demonstrated that the ER stress-mediated pathway is involved in the apoptosis.

Materials and methods Reagents and antibodies Ovalbumin (OVA) Grade V, arsenic trioxide, and aluminum hydroxide gel were purchased from Sigma Chemical Co (Poole, Dorset, UK). Mouse anti-CD4 microbeads and LS columns were purchased from Miltenyi Biotech (Bergisch Gladbach, Germany). GRP78-Ab, CHOP-siRNA and control siRNA were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). CHOP-Ab was purchased from ProteinTech Group, Inc (Chicago, IL). Annexin-VFITC Apoptosis Detection Kit was purchased from BD Bioscience (Palo Alto, CA). LipofectamineTM RNAiMAX was purchased from Invitrogen (Carlsbad, CA). Animals Female BALB/c mice aged 6–8 weeks were obtained from Slaikejingda Laboratory (Changsha, China), and maintained in the Animal Center of The Second Xiangya Hospital of Central South University. All animal studies were conducted in accordance with the principles and

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procedures outlined in the National Institutes of Health Guide for the Care and Use of Animals under assurance number A3873-1. All animal experimental protocols were approved by the Animal Subjects Committee of the Central South University, China. Ovalbumin sensitization and arsenic trioxide treatment Specific pathogen-free (SPF) grade mice were raised and kept on an OVA-free diet. We modified a previously established murine model of asthma. Female BALB/c mice, 6–8 weeks old, were sensitized and challenged with OVA as previously described [8]. Briefly, mice were sensitized by intraperitoneal injection with 10 lg OVA in 200 ll of PBS, and mixed with 2 mg alum on day 1 and 13. On days 19–24, mice were challenged with aerosolized OVA (5 % w/v) for 30 min once daily. ATO (2.5 mg/kg) was given by intraperitoneal injection 30 min before each OVA aerosol challenge. The mice were divided into three groups: the PBS group received PBS sensitization and PBS challenge, the OVA/PBS group received OVA sensitization, OVA challenge, and PBS treatment before each OVA aerosol challenge, and the OVA/ATO group received OVA sensitization, OVA challenge, and ATO treatment before each OVA aerosol challenge. Mice were killed 24 h after the last aerosol challenge (Fig. 1). Measurement of airway hyperresponsiveness Airway hyperresponsiveness was assessed 24 h after the final OVA challenge. Mice were intraperitoneally anesthetized with 500 mg/kg of 5 % chloral hydrate. The tracheas were exposed and cannulated for mechanical ventilation. Mice were mechanically ventilated with a small animal ventilator at a tidal volume of 10 ml/kg, and a frequency of 120 breaths/min. To examine the development of airway hyperresponsiveness, total airway resistance in response to increasing concentrations of methacholine (0.78, 1.56, 3.12, and 6.25 mg/ml) aerosol challenge were recorded using the Buxco pulmonary function apparatus. Airway resistance (RL) was obtained by measuring mouse airway flow and pressure. Bronchoalveolar lavage fluid (BALF) assessment To measure airway inflammation, we examined the accumulation of inflammatory cells in BALF. Mice were killed by intraperitoneal injection of chloral hydrate, and the trachea was cannulated and immediately lavaged three times with 1 ml saline. The lavage fluid was kept on ice and then centrifuged (4009g) at 4 °C for 10 min. BALF supernatant was stored at -80 °C. Total cell counts were counted in a counting-chamber. A differential count was

Ir J Med Sci Fig. 1 Mice model of asthma and treatment of ATO

performed on a smear and stained with hematoxylin–eosin stain solution. Histopathological analysis Mice lung tissues were fixed in 4 % polyformaldehyde solution at 4 °C overnight, and then embedded in paraffin. Paraffin was cut into 5-lm-thick sections. Sections were stained with hematoxylin–eosin (HE) or periodic acidschiff (PAS), and examined by light microscopy for histological changes. An inflammation score was assigned in a blinded fashion by a pathologist. Peribronchiolar and perivascular inflammation was scored as follows: 0, normal; 1, few cells; 2, a ring of inflammatory cells one cell layer deep; 3, a ring of inflammatory cells two to four cells deep; and 4, a ring of inflammatory cells of more than four cells deep. PAS scores were assigned by a blinded investigator examining 10 consecutive fields per slide as follows: 0, \5 % PAS-positive goblet cells; 1, 5–25 %; 2, 25–50 %; 3, 50–75 %; 4, [75 %, with 0 being negative and 1–4 being positive for PAS-staining bronchi. CD4? T cell isolation and culture Total CD4?T cells were purified from the spleen by positive selection, using immunomagnetic beads according to the manufacturer’s protocol (Miltenyi Biotech). Mononuclear cells separated from the spleen were incubated with CD4 (L3T4) MicroBeads (100 ll/1 9 108 cells) for 15 min at 4 °C in magnetic cell sorting (MACS) buffer. After two additional washes, the cell suspension was loaded onto a MACS column, which was placed in the magnetic field of a MACS separator. Positive cells containing CD4 antigen were harvested. The CD4? T cells (1 9 106 cells/ml) were grown in RPMI-1640 media containing 10 % fetal calf serum and antibiotics in the presence of ConA (5 lg/ml) with or without ATO. Detection of apoptosis in vitro Apoptosis measurements were done with Annexin-V/PI staining according to the manufacturer’s protocol. Briefly,

cells were harvested after being cultured with or without arsenic trioxide for 20 h. After two additional washes, the cells were incubated for 15 min with 5 lL Annexin-VFITC and 5 lL PI. Annexin-V?/PI-cells were characterized as apoptotic cells, as analyzed by flow cytometry. Western blotting CD4? T cells were harvested, followed by extraction of total protein using RIPA lysis buffer containing protease inhibitors (Beyotime, Shanghai, China). Cell lysates were centrifuged at 12,000 rpm at 4 °C for 5 min, and the supernatant was transferred to a fresh 0.5-mL tube. Protein concentration was measured using the Bradford protein assay Kit (Beyotime, Shanghai, China). An equal amount of protein was loaded onto 10 % SDS-PAGE gels. Proteins were electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane, blocked in 5 % skim milk in Tris-buffered saline/Tween buffer, and incubated overnight with the appropriate primary antibodies. Western blot analysis was performed with rabbit polyclonal antibody anti-GRP78 (1:1,000; Santa Cruz) and rabbit polyclonal antibody anti-CHOP (1:1,000; ProteinTech Group, Inc). The membrane was washed to remove unbound antibody, incubated with a 1:3,000 dilution of HRP-conjugated secondary antibody for 1 h, and washed again. Detection was carried out with the ECL Western blotting kit, after which the blots were exposed to an X-ray film. RNA interference Pre-designed siRNA against mice CHOP (sc-35438) and control scrambled siRNA (sc-37007) were purchased from Santa Cruz. The knockdown experiments with siRNA were carried out by incubating 1 9 105 CD4? T cells in 12-well plates overnight before transfection. Then, CD4? T cells were treated with siRNA duplexes (sc-35438) containing a pool of three target-specific 20–25 nt siRNAs designed to knockdown CHOP expression, in the presence of Lipofectamine RNAi max (Invitrogen), according to the manufacturer’s instruction. The final siRNA concentrations were 30 nM. CD4? T cells were then cultured in normal

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growth medium for 24 h. Knockdown of CHOP expression was confirmed by real-time PCR and Western blot analysis. Twenty-four hours after transfection, CD4?T cells were treated with or without arsenic trioxide for 20 h. Cells were then subjected to Annexin-V/PI staining for the apoptosis assay.

Statistical analysis All data are expressed as mean ± standard deviation (SD). For in vivo experiments, each group consisted of 4–6 mice. Significance (set at p \ 0.05) was assessed by one-way ANOVA or by non-parametric rank-based tests.

Real-time PCR Results Oligonucleotide primers for target genes were designed by Primer ExpressTM software, version 5.0 (Beijing Sunbiotech Co., Ltd. China.). Primers were designed for CHOP with the forward primer 50 -TGTTGAAGATGAGCGGGTGG-30 and the reverse primer 50 -CGTGGACCAGGTTCTGCTTT-30 ; b-actin was used as the endogenous control with forward primer 50 -GCATCTTGGGCTACACTGAGGA-30 and reverse primer 50 -GTGGGTGGTCCAGGGTTTCTTA-30 . Total RNA from CD4? T cells was isolated using Trizol reagent according to the manufacturer’s instructions (Invitrogen). First strand cDNA was synthesized with the RevertAid First Strand cDNA Synthesis Kit (Fermentas), using 5 lg of RNA per 12 ll reaction and oligo (dT) primer. Real-time PCR reactions were set up using the SYBR Green qRCR Mix protocol. PCR was performed according to the following conditions: denaturation at 95 °C for 5 min, 40 cycles of denaturation at 94 °C for 20 s, annealing at 57 °C for 20 s and extension at 72 °C for 20 s, followed by a final extension at 72 °C for 10 min. Three replicates were run for each gene per sample. The comparative threshold cycle (Ct) value method was used to analyze relative gene expression.

Arsenic trioxide treatment decreased airway hyperresponsiveness in mice To investigate the effect of arsenic trioxide on airway hyperresponsiveness in response to increasing concentrations of methacholine, we measured airway resistance in mechanically ventilated mice. As shown in Fig. 2, the OVA/PBS group had significant increases in airway reactivity in response to methacholine compared to the PBS group. This indicates that the murine model used in the present study exhibited airway hyperresponsiveness. In contrast, the OVA/ATO group showed significantly lower airway resistance than the OVA/PBS group. Thus, mice treated with arsenic trioxide had significantly decreased airway reactivity in response to methacholine. The number of inflammatory cells in bronchoalveolar lavage fluid of arsenic trioxide-treated mice significantly decreased Twenty-four hours after the last challenge, bronchoalveolar lavage fluid assessment was performed. We analyzed the inflammatory cells present in the bronchoalveolar lavage fluid of the experimental mice. Total cell counts and differential cell counts were determined. As show in Fig. 3, the number of total cells, eosinophils, lymphocytes, and neutrophils significantly increased in the OVA/PBS group compared to the PBS group, and the administration of

Fig. 2 Effect of ATO on airway responsiveness (AHR) to inhaled methacholine in mice. AHR was measured 24 h after the final challenge. AHR is expressed as percentage change from the baseline level of airway resistance (RL % of baseline). Data are graphed as mean ± SD; n = 4–6; *p \ 0.05 for the OVA/PBS group vs. PBS group, #p \ 0.05 for OVA/ATO group vs. OVA/PBS group. The PBS group only received PBS sensitization and PBS challenge. The OVA/ PBS group received OVA sensitization, OVA challenge, and PBS treatment before each OVA aerosol challenge. The OVA/ATO group received OVA sensitization, OVA challenge, and ATO treatment before each OVA aerosol challenge

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Fig. 3 Effect of ATO on preventing inflammatory cell infiltration in the airway. Twenty-four hours after the last challenge, BALF was assessed and the lavage was analyzed for changes in total number of cells, macrophages, lymphocytes, eosinophils, and neutrophils. Each column represents the mean ± SD, for n = 4–6, at the respective column, *p \ 0.05 for OVA/PBS group vs. PBS group, #p \ 0.05 for OVA/ATO group vs. OVA/PBS group

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Fig. 4 ATO treatment alleviated lung inflammation. Mice were killed by intraperitoneal injection of chloral hydrate 24 h after the last challenge. Histopathological changes in the lungs of different groups were detected by HE or PAS stain. a PBS group (9100); b OVA/PBS group (9100); c OVA/ATO group (9100); d PBS group (9100); e OVA/PBS group (9100); f. OVA/ATO group (9100). Quantitative

analyses of inflammatory cell infiltration and mucus production were evaluated in lung sections with a method described in ‘‘Materials and methods’’. The comparisons of HE and PAS staining was carried out with non-parametric rank-based tests. n = 4–6, at the respective column, *p \ 0.05 for OVA/PBS group vs. PBS group, #p \ 0.05 for OVA/ATO group vs. OVA/PBS group

arsenic trioxide decreased total inflammatory and differential cell counts compared to the OVA/PBS group. Thus, the number of inflammatory cells in the bronchoalveolar lavage fluid of arsenic trioxide-treated mice significantly decreased.

cells by flow cytometry. Freshly isolated CD4? T cells were harvested and stained with Annexin-V/PI to detect the percentage of apoptotic cells by flow cytometry. As shown in Fig. 5, Annexin-V?/PI-cells, which are defined as the early phase of apoptotic cells, were notably increased in arsenic trioxide-treated mice compared to untreated mice (Fig. 5a, b). To investigate the effect of arsenic trioxide on promoting apoptosis of CD4? T cells in vitro, we isolated and cultured primary spleen CD4? T cells from asthmatic mice. CD4? T cells were treated with different concentrations of arsenic trioxide for 20 h. The percentage of apoptotic cells was detected by flow cytometry, and the optimal concentration of arsenic trioxide was determined by assessing apoptosis. The data shown in Fig. 5c, d indicate that the apoptosis of CD4? T cells increased when cultured with arsenic trioxide compared to cells cultured without arsenic trioxide.

Arsenic trioxide treatment alleviated inflammation of the lung Histological examination revealed marked inflammation of the lungs in the OVA/PBS group mice; many inflammatory cells infiltrated into the subepithelial and mucus hyperproduction by goblet cells within the bronchi when compared to mice in the PBS group (Fig. 4). However, inflammatory cell infiltration in arsenic trioxide-treated mice was much less. Arsenic trioxide treatment promoted apoptosis of CD4? T cells in vivo and in vitro To determine if administration of arsenic trioxide by intraperitoneal injection can induce apoptosis of CD4? T cells in asthmatic mice, we analyzed apoptosis of CD4? T

Arsenic trioxide treatment increased expression of GRP78 and CHOP in CD4? T cells Arsenic trioxide can induce apoptosis through the ER stress pathway. CHOP is a specific transcription factor involved

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Ir J Med Sci Fig. 5 ATO treatment promoted CD4? T cell apoptosis cells in vivo and in vitro. Mice were killed by intraperitoneal injection of chloralic hydras 24 h after the final OVA challenge. Then, CD4? T cells were obtained by MACS isolation. Freshly isolated CD4? T cells (1 9 106 cells/ml) were cultured with RPMI-1640 media containing 10 % fetal calf serum and antibiotics in the presence of ConA (5 lg/ml) for 24 h. CD4? T cells were harvested and stained with Annexin-V/PI to detect the percentage of apoptotic cells (a, b). Primary spleen CD4? T cells were isolated from asthmatic mice that only received OVA sensitization and OVA challenge without any treatment. The CD4? T cells were then cultured with different concentrations of ATO in RPMI-1640 media containing 10 % fetal calf serum and antibiotics in the presence of ConA for 20 h. CD4? T cells were harvested and stained with Annexin-V/PI to detect the percentage of apoptotic cells (c, d). Annexin-V?/PI- cells were characterized as apoptotic cells. Data are graphed as mean ± SD, for n = 3; *p \ 0.05 for OVA/PBS group vs. PBS group (b), or for 3 vs. 0 lM; 5 vs. 0 lM (d). #p \ 0.05 for OVA/ATO group vs. OVA/PBS group (0 lM group contains an equivalent amount of solvent)

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inhibited CHOP mRNA expression in CD4? T cells (Fig. 7a). The siRNA also inhibited CHOP protein production in CD4? T cells (Fig. 7b). Next, we examined whether the reduction of CHOP protein would have any effect on apoptotic induction. We found that cells that were treated with CHOP-siRNA were less sensitive to apoptotic induction by arsenic trioxide compared to cells treated with control siRNA (Fig. 7c). The siRNA also downregulated expression of CHOP induced by arsenic trioxide (Fig. 7d). These results indicate that CHOP plays a significant role in the induction of apoptosis by arsenic trioxide in CD4? T cells.

Discussion

Fig. 6 ATO treatment increased expression of GRP78 and CHOP in CD4? T cells. CD4? T cells were isolated from the PBS, OVA/PBS, or OVA/ATO groups. Then, CD4? T cells (1 9 106 cells/ml) were cultured with RPMI-1640 media containing 10 % fetal calf serum and antibiotics in the presence of ConA for 24 h. CD4? T cells were harvested, and cell lysates were prepared for detection of CHOP expression (a). CD4? T cells isolated from only OVA-sensitized and challenged mice were incubated with ATO (5 lM) for different time intervals, and the expression of GRP78 and CHOP were examined by Western blotting (b, c)

in ER stress, and overexpression of this factor can induce apoptosis. To further explore if the administration of arsenic trioxide by intraperitoneal injection in vivo can effect CHOP expression, we measured CHOP protein levels in the cellular lysates of CD4? T cells (Fig. 6a). We found that administration of arsenic trioxide upregulated CHOP expression in CD4? T cells. We also investigated the expression of GRP78 and CHOP in CD4? T cells, which were cultured with arsenic trioxide in vitro. The data (Fig. 6b, c) showed that arsenic trioxide markedly increased the levels of GRP78 and CHOP, especially after CD4? T cells were treated with arsenic trioxide for 5 h. Knockdown of CHOP by siRNA decreased apoptosis of CD4? T cells cultured with arsenic trioxide Our results indicate that arsenic trioxide induces the upregulation of CHOP, resulting in apoptotic induction. We studied the underlying mechanism of CHOP-induced apoptosis by employing siRNA duplexes, which selectively

This study found that arsenic trioxide treatment alleviated airway hyperresponsiveness and reduced inflammation of the lung in a murine model of asthma. Furthermore, arsenic trioxide treatment promoted apoptosis of CD4? T cells, which involved the ER stress–CHOP pathway. Although arsenic trioxide is often considered a toxicant, it is currently used in the clinic for treating cancer. Arsenic trioxide is a powerful ancient medication for a variety of illness with the principle of ‘‘using a toxic substance against another toxic substance’’ in traditional Chinese medicine. Recently, it was found that arsenic trioxide treatment had an amazing effect on relieving lymphoproliferative and autoimmune syndrome in MRL/lpr mice [9]. Kuan-Hua Chu et al. demonstrated that intraperitoneal injection of arsenic trioxide (2.5 mg/kg) alleviates airway hyperresponsiveness in a murine model of asthma. Data from the current study is in accordance with those results. Specifically, we found that in a murine model of asthma, total airway resistance of arsenic trioxide-treated mice was lower than in untreated mice. Moreover, similar to other reports [10], arsenic trioxide exerted its anti-inflammatory action in a murine model of asthma. Arsenic trioxide is considered a toxicant, but the dosage we used in the experiment (2.5 mg/kg) exhibited little toxicity. Activated CD4? T cells in the peripheral blood and airway tissues is an invariant feature of asthma [11]. Some reports have suggested that T cells from asthmatic patients, in comparison to those from healthy controls, exhibit decelerated spontaneous apoptosis after a 24 h incubation in vitro, and arsenic trioxide treatment in vitro significantly increases the percentage of apoptotic T cells, which was isolated from asthmatic patients [12]. In our study, we found that the spontaneous apoptosis of CD4? T cells isolated from OVA/PBS group mice was decelerated compared to the PBS group. Furthermore, our study revealed that administration of arsenic trioxide in vivo increased the spontaneous apoptosis of CD4? T cells in a

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Ir J Med Sci Fig. 7 Knockdown of CHOP by siRNA decreased the apoptosis of CD4? T cells cultured with ATO. Cells were transfected with control scrambled siRNA and siRNA against mice CHOP for 24 h. The mRNA and protein expression of CHOP was detected by real-time PCR and Western blotting, respectively (a, b). After siRNA transfection the CD4? T cells were incubated with ATO (5 lM) for 20 h, and the percentage of apoptotic cells was then determined by flow cytometric analysis of Annexin-V/PI double staining (n = 3) (c). In addition, after transfection with siRNA against mice CHOP or control scrambled siRNA for 24 h, CD4? T cells were incubated with ATO (5 lM) for 5 h, and the expression of CHOP was determined by Western blotting (d). Results are expressed as the mean ± SD; n = 3,*p \ 0.05 compared with control

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murine model of asthma. The apoptotic rate increased when CD4? T cells were cultured with arsenic trioxide in vitro. Therefore, the data from our study indicate that arsenic trioxide treatment can promote the apoptosis of CD4? T cells both in vivo and in vitro. Arsenic trioxide induced cell apoptosis in cultured osteoblasts after 24 h, through endoplasmic reticulum stress [13], although clear ER stress was induced after human neutrophils cultured with arsenic trioxide for 20 h. The ER network was perturbed in arsenic trioxide-treatment cells as vacuoles were observed in arsenic trioxideinduced cells but not in fresh cells [14]. When T cells were cultured with arsenic trioxide for 24 h, their apoptosis rate increased. In this study, arsenic trioxide induced apoptosis in CD4? T cells after being cultured for 20 h. Arsenic trioxide largely effects asthma. Eotaxin is an eosinophil-selective chemoattractant that has been identified as a potent activator of eosinophils, inducing eosinophils to generate superoxide and release granule proteins. Eosinophils are closely related to airway hyperresponsiveness. Kuan-Hua Chu et al. [5] demonstrated that arsenic trioxide directly inhibited the secretion of eotaxin by lung epithelial cells, resulting in decreased numbers of eosinophils recruited into the airway. Therefore, the inhibitory effect of arsenic trioxide on airway hyperresponsiveness may be partially attributed to the inhibitory effect of arsenic trioxide on eotaxin. On the other hand, arsenic trioxide promoted eosinophils apoptosis [15]. This effect can also alleviate airway hyperresponsiveness. Lin-Fu Zhou et al. [10] demonstrated that arsenic trioxide can inhibit the activation of NF-jB and abrogate allergeninduced inflammation. In our study, we found that arsenic trioxide treatment increased the spontaneous apoptosis of CD4? T cells. The effect of arsenic trioxide on the apoptosis of CD4? T cells partially contribute to its effect on alleviating airway hyperresponsiveness and reducing inflammation. In this study we found that arsenic trioxide can induce apoptosis of CD4? T cells. Interleukin-2 (IL-2) was discovered in 1976 as a T cell growth factor activity in the supernatants of activated T cells [16]. IL-2 is a pleiotropic cytokine that drives T cell growth, which maintains the viability of IL-2-dependent cells. The cloned murine cytotoxic T cell line CT6 solely requires IL-2 for viability and cell cycle progression. IL-2 is produced primarily by CD4? T cells following their activation by antigen. A recent study discovered the complex roles of IL-2 in broadly modulating T cells for T helper cell differentiation. It was found that IL-2 can prime and potentially maintain Th1 and Th2 differentiation and expand the populations of such cells, whereas it inhibits Th17 differentiation but also expands Th17 cells [17]. The in vivo study in mice showed that treatment with combined arsenic trioxide and anti-

CD154/LFA-1 significantly reduce the production of IL-2 [18]. Further research on the effect of arsenic trioxide on the production of IL-2 in CD4? T cells may lead to profound discoveries. It is known that there are three different signaling pathways that lead to apoptosis: the extrinsic death receptor-dependent pathway, the intrinsic mitochondria-dependent pathway, and the intrinsic endoplasmic reticulum (ER) stress-mediated pathway. The efficient function of the ER is essential for proper cellular activities and survival. Under certain circumstances, biochemical and physiological stimuli interfere with ER function, causing the accumulation and aggregation of unfolded or misfolded proteins in the ER lumen [19]. The ER can sense this stress and in turn, activates compensatory mechanisms, which are termed the ER stress response [20]. Unfolded protein response (UPR) is just one of the compensatory mechanisms that are activated. GRP78, a central mediator of ER homeostasis, is considered to be the primary sensor of ER stress. Under normal conditions, GRP78 binds to each of the ER stress transducers, including PKR-like ER kinase (PERK), inositol-requiring enzyme 1 (IRE1), and activating transcription factor 6 (ATF6), all of which repress UPR signaling pathways. Under ER stress, the accumulation of unfolded or misfolded proteins dissociates GRP78 from ER stress transducers, leading to activation of these transducers. The activation of IRE1 and ATF6 signaling promotes expression of the pro-apoptotic transcription factor CHOP. Activated PERK phosphorylates eIF2a (eukaryotic initiation factor) and promotes translation of ATF4. Then, ATF4 upregulates the expression of CHOP. GRP78 is one of the key markers of ER stress. When biochemical and physiological stimuli induce ER stress, the expression of GRP78 increases. Several studies suggest that arsenic trioxide promotes apoptosis in lens epithelial cells, osteoblasts, and neutrophils through the ER stress-mediated pathway. Thus, we were interested in determining if arsenic trioxide induces ER stress-related events in CD4? T cells as well. We found that culturing CD4? T cells with arsenic trioxide (5 lM) can increase the expression of GRP78 and CHOP. Therefore, we conclude that arsenic trioxide induces activation of the ER stress response, and promotes apoptosis in CD4? T cells. CHOP is a transcription factor belonging to the C/EBP family [21]. It is an important element of the switch from pro-survival to pro-death signaling. The transcription of CHOP is strongly induced in response to ER stress, leading to the induction of apoptosis in a variety of cell types. CHOP is a pro-apoptotic protein that is able to downregulate the expression of Bcl-2, and upregulate the expression of some pro-apoptotic members of the Bcl-2 family [22, 23]. Several studies in Chop-/- mouse revealed that CHOP deficiency provides partial resistance

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to ER stress-induced apoptosis [24, 25]. In this study, we knocked down the expression of CHOP using a siRNA duplex against CHOP mRNA, and found that inhibition of CHOP attenuated arsenic trioxide-induced apoptosis in CD4? T cells. This result indicates that the ER stress– CHOP pathway is therefore involved in arsenic trioxideinduced apoptosis of CD4? T cells. This study found that arsenic trioxide increased CD4? T cells apoptosis by nearly 14 % compared to no treatment (Fig. 5). As can be seen in Fig. 7, CHOP-siRNA reduced these effects to nearly 7 %. Arsenic trioxide acts as an inducer of apoptosis through different pathways. For example, arsenic trioxide induced apoptosis of breast cancer cells, not only through inactivation of Notch signaling pathway, but also can through activation of caspase3 [26, 27]. Arsenic trioxide induces apoptosis of Burkitt lymphoma cell lines through multiple apoptotic pathways, such as arresting the cell cycle, decreasing the respiratory function and transmembrane potential of mitochondrial, and downregulating the expressions of Survivin, Bcl-2, MCL-1, and VEGF [28]. Therefore, the apoptosis of CD4? T cells induced by arsenic trioxide may involve another signaling in addition to ER stress–CHOP pathway. These other potential signaling pathways may explain the differences observed between Figs. 5 and 7. Together, our research indicates that arsenic trioxide may have therapeutic potential in the treatment of asthma.

Conclusion These data demonstrated that arsenic trioxide can reduce the severity of asthma attacks and induce the apoptosis of CD4? T cells which the ER stress–CHOP pathway involved. Acknowledgments This study was supported by the Respiratory Department and Central Laboratory at the Second Xiangya Hospital of Central South University. We are very grateful to Jun-wei Deng, You-wen Liu, and Xi ‘nian Jiang for their technical assistance. And we thank for the assistance from the key project foundation of The Project of Science and Technology of Hunan Province (No. 2010FJ2004). Conflict of interest peting interests.

The authors declare that they have no com-

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