Signaling Pathway Involved in the Immunomodulatory Effect of

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Signaling Pathway Involved in the Immunomodulatory Effect of Ganoderma atrum Polysaccharide in Spleen Lymphocytes Qiang Yu, Shao-Ping Nie,* Jun-Qiao Wang, Dan-Fei Huang, Wen-Juan Li, and Ming-Yong Xie* State Key Laboratory of Food Science and Technology, Nanchang University, 235 Nanjing East Road, Nanchang, Jiangxi 330047, People’s Republic of China ABSTRACT: The aim of this study was to investigate the molecular mechanism underlying the immunomodulatory effect of Ganoderma atrum polysaccharide (PSG-1) in spleen lymphocytes. Our results showed that PSG-1 increased the intracellular Ca2+ concentration and calcineurin (CaN) activity. Moreover, PSG-1 was found to elevate nuclear factor of activated T cells (NFAT) activity, but this effect could be diminished by the treatment of CaN inhibitors (cyclosporin A and FK506). PSG-1-induced interleukin (IL)-2 production was also inhibited by cyclosporin A and FK506. In addition, PSG-1 was found to significantly enhance protein kinase C (PKC) activity. PKC was involved in induction of NFAT activity by PSG-1, as evidenced by abrogation of NFAT activity by PKC inhibitor calphostin C, which significantly decreased PSG-1-induced IL-2 production. On the basis of these results, we concluded that PSG-1 may induce activation of spleen lymphocytes at least in part via the Ca2+/CaN/NFAT/ IL-2 signaling pathway and the PKC/NFAT/IL-2 signaling pathway cooperatively regulated PSG-1-induced activation of spleen lymphocytes. KEYWORDS: Ganoderma atrum polysaccharide, spleen lymphocytes, immunomodulatory effect, signaling pathway, interleukin-2

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INTRODUCTION Lymphocytes are the major cellular components of the adaptive immune response. Lymphocytes are activated after a process known as antigen presentation by antigen-presenting cells. The activation of lymphocytes is a complex signaling transduction process that involves various secondary messengers and signal molecules, such as Ca2+, calcineurin (CaN), protein kinase C (PKC), and nuclear factor of activated T cells (NFAT).1 Ca2+ is an universal secondary messenger in almost all immune cells.2 Ca2+ signals play pivotal roles in diverse cellular functions, including proliferation, differentiation, and gene transcription.3 An increase in intracellular Ca2+ signals leads to activation of the serine/threonine phosphatase CaN, which is a calmodulin-dependent enzyme and has crucial functions in neurons, cardiac and skeletal muscle cells, and lymphocytes.4 NFAT is a family of four transcription factors (NFATc1, NFATc2, NFATc3, and NFATc4), which are first described in lymphocytes. NFAT is critical for an effective immune response because it mediates transcription of a large number of cytokine genes.5 Besides, NFAT is known as a key substrate of CaN. Normally, NFAT resides in the cytoplasm of resting cells in a phosphorylated form. In response to the rise of the intracellular Ca2+ level, activated CaN dephosphorylates NFAT, which thereby translocates to the nucleus and binds to the promoter and, in turn, initiates the transcription of specific cytokine genes.6 PKC, a phospholipid-dependent serine/threonine kinase, appears to participate in the signal transduction pathways of diverse cells.7 In lymphocytes, upon stimulation and subsequent Ca2+ signal mobilization, inactive PKC is promoted to translocate from the cytosol to the plasma membrane for attachment and activation.8 Polysaccharides from fungus, plant, and other natural sources that serve as an immunomodulating agent without toxicity and © XXXX American Chemical Society

significant side effects have been attracting more and more attention.9−11 Mounting studies have reported that polysaccharides possess an immunomodulatory effect on lymphocytes.12,13 Ganoderma, well-known as “Lingzhi” in Chinese, has been safely used as an ingredient of traditional medicine and functional food for thousands of years in oriental countries.14 Ganoderma atrum is one of the most well-known mushrooms of Ganodermataceae, with a great deal of interest.15 We recently isolated and purified a polysaccharide from G. atrum, named as PSG-1, with a purity of >99.8%, whose primary structural features and molecular weight were characterized.16,17 Our previous studies have shown that PSG-1 possesses a variety of biological functions, including antitumor,18−21 immunomodulatory,22−24 cardiovascular protection,25 chemoprotective,26 and hypoglycemic27 activities. However, the molecular mechanism underlying the immunomodulatory effect of PSG-1 in spleen lymphocytes has not been investigated. Therefore, the present study was designed to elucidate the signaling pathway involved in the activation of spleen lymphocytes in response to PSG-1.

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MATERIALS AND METHODS

Materials and Reagents. Enzyme linked immunosorbent assay (ELISA) kits were from R&D Systems (Minneapolis, MN). Fluo-3/ AM was purchased from Molecular Probes, Inc. (Eugene, OR). The CaN assay kit was from Genmed Scientifics, Inc. (Shanghai, China). pGL4.30[luc2P/NFAT-RE/Hygro], FuGENE HD transfection reagent, luciferase assay system, and PepTag assay for non-radioactive detection of PKC kit were purchased from Promega (Madison, WI). Received: January 3, 2015 Revised: February 25, 2015 Accepted: February 25, 2015

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DOI: 10.1021/acs.jafc.5b00028 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 1. Effect of PSG-1 on the intracellular Ca2+ concentration in spleen lymphocytes. Spleen lymphocytes were seeded at a density of 1 × 106 cells/well in the 24 well plate and incubated with PSG-1 (20, 40, 80, and 160 μg/mL) for 48 h. The cells were washed twice with PBS and incubated with the Fluo-3/AM at 37 °C for 60 min. Flow cytometry was performed to measure the fluorescent signal. by incubation with the fluorescent probe Fluo-3/AM at 37 °C for 60 min. After washing twice with PBS, the [Ca2+]i level, seen as the fluorescent signal, was measured by flow cytometry. Measurement of CaN Activity. Spleen lymphocytes were seeded at a density of 1 × 106 cells/well in the 24 well plate and stimulated with various concentrations of PSG-1 for 48 h. CaN activity was measured using a CaN activity assay kit according to the instructions of the manufacturer. Briefly, the cells were treated by lysis buffer (reagent B) provided in the kit. The RII phosphopetide was used as the substrate for CaN. The detection of free inorganic phosphate released from RII by CaN was based on the ferrous sulfate method. The optical density at 660 nm was determined for each sample using a microplate reader (Bio-Rad, Hercules, CA). The specific CaN activity was expressed as the fold induction over untreated cells. Transient Transfection and Luciferase Reporter Assay. Spleen lymphocytes were transiently transfected with pGL4.30[luc2P/NFAT-RE/Hygro] using FuGENE HD transfection reagent according to the instructions of the manufacturer. Transfected cells were subcultured and seeded at 2 × 105 cells/well in the 24 well plate. The cells were incubated with various concentrations of PSG-1 for 48 h. After incubation, the cells were washed with PBS and extracted in lysis buffer. Luciferase activity was measured using the luciferase assay system according to the instructions of the manufacturer. The specific transactivation was expressed as the fold induction over untreated cells.

Cell culture products were obtained from Life Technologies (Paisley, U.K.). Preparation of Spleen Lymphocytes. Female BALB/c mice (8 weeks old, 18−20 g) were purchased from Shanghai Slac Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). The extirpated spleens were minced into small pieces with scissors in a germ-free condition and then suspended in RPMI 1640 medium. Single-cell suspension was prepared by filtering the suspension through a sterile sieve mesh. The cells were treated with lysis buffer (0.15 M NH4Cl, 0.01 M KHCO3, and 0.1 mM Na2EDTA at pH 7.4) to remove red blood cells, followed by washing twice with cold phosphate-buffered saline (PBS). Then, the cells were adjusted to the concentration of 5 × 106 cells/mL in RPMI 1640 medium supplemented with 10% fetal calf serum and incubated for 3 h in Petri dishes. The suspended cells were collected. Cytokine Measurement by ELISA. Spleen lymphocytes were seeded at a density of 5 × 105 cells/well in the 96 well plate and cultured with various concentrations of PSG-1 for 48 h. The supernatants were harvested, and a level of IL-2 was determined by an ELISA kit according to the instructions of the manufacturer. Measurement of Intracellular Ca2+. [Ca2+]i was measured in spleen lymphocyte using a Fluo-3/AM fluorescent probe. Briefly, spleen lymphocytes were seeded at a density of 1 × 106 cells/well in the 24 well plate and stimulated with various concentrations of PSG-1 for 48 h. The cells were collected and washed twice with PBS, followed B

DOI: 10.1021/acs.jafc.5b00028 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Assay of PKC Activity. Spleen lymphocytes were seeded at a density of 2 × 106 cells/well in the 6 well plate and stimulated with various concentrations of PSG-1 for 48 h. Then, the cells were collected and washed twice with PBS. The PKC activity was measured using the PepTag assay for non-radioactive detection of PKC kit according to the instructions of the manufacturer. Briefly, the cells were resuspended and homogenized in 0.5 mL of cold PKC extraction buffer and then centrifuged at 14000g for 5 min, and the supernatants were purified on a 1 mL column of DEAE cellulose. The resultant fractions were eluted by PKC extraction buffer and incubated with PKC reaction mixture, followed by agarose gel separation. The bands were quantization by spectrophotometry. Statistical Analysis. Values are expressed as means ± standard error of the mean (SEM). One-way analysis of variance followed by the Student−Newman−Keuls test was used to determine the statistical significance between various groups. A value of p < 0.05 was considered to be statistically significant.

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Figure 3. Effect of PSG-1 on NFAT transactivation activity in spleen lymphocytes. Spleen lymphocytes were transiently transfected with pGL4.30[luc2P/NFAT-RE/Hygro] and seeded at 2 × 105 cells/well in the 24 well plate. The cells were stimulated with various concentrations of PSG-1 for 48 h. After incubation, the cells were washed and extracted in lysis buffer. Then, luciferase activity was measured using the luciferase assay system according to the instructions of the manufacturer. The data are expressed as the mean ± SEM for three separate experiments. (∗) p < 0.05 and (∗∗) p < 0.01 versus the control group.

RESULTS Effect of PSG-1 on the Ca2+ Concentration in Spleen Lymphocytes. To examine the effect of PSG-1 on the Ca2+ concentration in spleen lymphocytes, flow cytometry was performed to detect the intracellular Ca2+ concentration after treatment with various concentrations of PSG-1 (20, 40, 80, and 160 μg/mL). As shown in Figure 1, PSG-1 administration elevated the Ca2+ concentration in a dose-dependent manner. Effect of PSG-1 on CaN Activity in Spleen Lymphocytes. The colorimetric method was used to measure the CaN activity in spleen lymphocytes. It was found that PSG-1 (20, 40, 80, and 160 μg/mL) dose-dependently enhanced CaN activity in spleen lymphocytes (Figure 2).

dose-dependent manner, which suggested that PSG-1 could enhance NFAT transactivation activity in spleen lymphocytes. Effect of CaN Inhibitors on NFAT Transactivation Activity Induced by PSG-1 in Spleen Lymphocytes. To determine the role of CaN in PSG-1-mediated activation of NFAT, we used the specific pharmacological antagonists cyclosporin A (CsA) and FK506, which inhibit the activation of CaN. Working concentrations of the inhibitors did not affect the viability of cells (data not shown). Transfected spleen lymphocytes were pre-incubated with various concentrations of CaN inhibitors, CsA (0.25, 0.5, and 1 μM) and FK506 (0.25, 0.5, and 1 μM) for 30 min and then treated with PSG-1 (160 μg/mL) for an additional 48 h. As shown in Figure 4A, luciferase activity was gradually reduced by pretreatment with increasing concentrations of CsA. Similarly, there was a dosedependent decrease of luciferase activity by the pretreatment of FK506, and PSG-1-induced luciferase activity returned to the basal level when pretreated with 1 μM FK506. Effect of CaN Inhibitors on IL-2 Production Induced by PSG-1 in Spleen Lymphocytes. To examine the potential role of CaN in the regulation of IL-2 production in PSG-1-stimulated spleen lymphocytes, the cells were pretreated by CsA (0.25, 0.5, and 1 μM) and FK506 (0.25, 0.5, and 1 μM) for 30 min, followed by PSG-1 treatment (160 μg/mL) for 48 h, and then, the IL-2 level was measured by ELISA. As shown in Figure 4B, CsA and FK506 dose-dependently inhibited the PSG-1-induced IL-2 production in spleen lymphocytes. Effect of PSG-1 on PKC Activity in Spleen Lymphocytes. Furthermore, the effect of PSG-1 on PKC activity was measured. PKC activity was dramatically increased by PSG-1 (20, 40, 80, and 160 μg/mL) in spleen lymphocytes, as compared to the control group (Figure 5). Effect of the PKC Inhibitor on NFAT Transactivation Activity Induced by PSG-1 in Spleen Lymphocytes. To investigate the role of PKC on NFAT activation induced by PSG-1 in spleen lymphocytes, PKC inhibitor calphostin C (25, 50, and 100 nM) was used to treat the transfected spleen

Figure 2. Effect of PSG-1 on CaN activity in spleen lymphocytes. Spleen lymphocytes (2 × 106 cells/well) were seeded in the 6 well plate and cultured with various concentrations of PSG-1 for 48 h. The cells were harvested, and CaN activity was determined by the CaN activity assay kit according to the instructions of the manufacturer. The data are expressed as the mean ± SEM for three separate experiments. (∗) p < 0.05 and (∗∗) p < 0.01 versus the control group.

Effect of PSG-1 on NFAT Transactivation Activity in Spleen Lymphocytes. NFAT is known as an important transcription factor. In the present study, a transient transfection and luciferase reporter assay was used to determine the effect of PSG-1 on NFAT transactivation activity in spleen lymphocytes. As shown in Figure 3, PSG-1 treatment (20, 40, 80, and 160 μg/mL) robustly increased luciferase activity in a C

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Figure 5. Effect of PSG-1 on PKC activity in spleen lymphocytes. Spleen lymphocytes were seeded at a density of 2 × 106 cells/well in the 6 well plate and stimulated with various concentrations of PSG-1 for 48 h. The cells were collected for measuring PKC activity using the PepTag assay for non-radioactive detection of PKC kit according to the instructions of the manufacturer. The data are expressed as the mean ± SEM for three separate experiments. (∗) p < 0.05 and (∗∗) p < 0.01 versus the control group.

and 100 nM). ELISA was used to measure the IL-2 secretion in the supernatant. As shown in Figure 6B, the production of PSG-1-induced IL-2 was significantly decreased by all inhibitor treatments in a dose-dependent manner (p < 0.05). These results demonstrated that the PKC-related signaling pathway was involved in PSG-1-induced IL-2 secretion in spleen lymphocytes.

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DISCUSSION Cytokines are intercellular signaling proteins that exert profound effects on the control of homeostasis in the whole organism; thus, monitoring the level of cytokine production is a widely used method to evaluate augmentation activity on immune response. IL-2 is an important cytokine in the cellular and humoral responses. It plays a vital role in regulating functions of all subsets of T cells as well as NK cells, LAK cells, B cells, etc.28 Our previous study found that PSG-1 significantly increased IL-2 production in spleen lymphocytes, indicating that PSG-1 could activate spleen lymphocytes. To further elucidate the underlying molecular mechanism, we focused on characterizing the signaling pathways involved in the spleen lymphocyte activation by PSG-1. Ca2+ is known as the most widely used intracellular messenger, which encodes a range of cellular information through regulation of Ca2+ signals.2 In response to diverse stimuli, elevation in the concentration of Ca2+ in the cytosol triggers many types of events, which play essential roles in lymphocyte activation and maturation.29 In the present study, flow cytometry analysis using a Fura-3/AM fluorescence probe showed that PSG-1 dramatically increased the intracellular Ca2+ level in spleen lymphocytes. CaN is well-known as an unique Ca2+-dependent serine/threonine protein phosphatase, which was originally identified as a calmodulin-binding protein in the brain.30 It has been reported that CaN activity induced by the increased intracellular Ca2+ level played a critical role in diverse biological processes, including lymphocyte function.31 In our study, we found that CaN activity was noticeably enhanced by PSG-1 treatment, indicating that PSG-1 may activate spleen lymphocytes via the Ca2+/CaN pathway.

Figure 4. Effect of CaN inhibitors CsA and FK506 on NFAT transactivation activity and IL-2 production induced by PSG-1 in spleen lymphocytes. (A) Transfected spleen lymphocytes were preincubated with CsA (0.25, 0.5, and 1 μM) and FK506 (0.25, 0.5, and 1 μM) for 30 min and treated with PSG-1 (160 μg/mL) for an additional 48 h. Then, luciferase activity was measured. (B) Cells were pre-incubated with various concentrations of inhibitors for 30 min, followed by PSG-1 stimulation (160 μg/mL) for an additional 48 h. After incubation, released IL-2 in the supernatant was measured by ELISA. The data are expressed as the mean ± SEM for three separate experiments. (#) p < 0.05 versus the control group, and (∗) p < 0.05 versus the PSG-1 alone group.

lymphocytes for 30 min before PSG-1 treatment (160 μg/mL) for 48 h and then NFAT transactivation activity was measured. As shown in Figure 6A, in comparison to the PSG-1 group, luciferase activities in all three calphostin C groups (25, 50, and 100 nM) were obviously decreased. Effect of the PKC Inhibitor on IL-2 Production Induced by PSG-1 in Spleen Lymphocytes. The effect of PKC on PSG-1-induced IL-2 production was studied. Prior to the addition of PSG-1 (160 μg/mL), spleen lymphocytes were treated with different concentrations of PKC inhibitor (25, 50, D

DOI: 10.1021/acs.jafc.5b00028 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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used a pharmacological assay using specific CaN inhibitors (CsA and FK506) to investigate whether NFAT activation by PSG-1 was triggered by CaN. PSG-1-induced NFAT activity was found to be abrogated by the treatment of CsA and FK506. These results suggested that the Ca2+/CaN signaling pathway may act upstream of NFAT activation in PSG-1-stimulated spleen lymphocytes. It is well-documented that NFAT plays an essential role in immune response via mediating transcription of a number of cytokines. After translocation from the cytoplasm to the nucleus, NFAT subsequently binds to the promoter of cytokines and then stimulates gene expression.6 To determine the role of the Ca2+/CaN/NFAT pathway in the regulation of PSG-1-induced IL-2 production in spleen lymphocytes, we tested the effect of CaN inhibitors, CsA and FK506, on IL-2 secretion and found that IL-2 production was inhibited by CsA and FK506 in a dose-dependent manner. These results demonstrated that the Ca2+/CaN/NFAT pathway was involved in PSG-1-induced IL-2 secretion in spleen lymphocytes. Taken together, the above results illustrated that PSG-1 may induce spleen lymphocyte activation at least in part via the Ca2+/CaN/ NFAT/IL-2 signaling pathway. PKC, a calcium-dependent kinase, exists in many different cell types.7 PKC-regulated signaling pathways play significant roles in a wide variety of aspects of immune responses, including development, differentiation, activation, and survival of lymphocytes.8 Our results showed that PSG-1 significantly enhanced PKC activity in spleen lymphocytes, indicating the involvement of PKC in PSG-1-mediated spleen lymphocyte activation. More and more evidence indicate that PKC signaling also involves the regulation of NFAT activation. In fact, DNA binding by NFAT is quite weak; therefore, it needs a partner for tight association with DNA. Thus, Ca2+/CaN signaling becomes dependent upon coincident PKC signaling for activation of NFAT-dependent transcription.34,35 To study the putative role of PKC in PSG-1-induced NFAT activation, we tested the effect of the PKC inhibitor on NFAT activity and found that inhibition of PKC by calphostin C diminished PSG1-induced NFAT activity in a dose-dependent manner, suggesting that the induction of NFAT activity by PSG-1 was dependent upon PKC in spleen lymphocytes. Furthermore, we examined whether PKC was involved in the regulation of IL-2 production induced by PSG-1. Pharmacological dissection using specific PKC inhibitor calphostin C significantly decreased the PSG-1-induced IL-2 production, indicating that PKC was involved in IL-2 production stimulated by PSG-1. On the basis of these results, we concluded that the PKC/NFAT/ IL-2 signaling pathway may play an important role in the lymphocyte activation by PSG-1. In summary, the present study established a signaling pathway, Ca2+/CaN/NFAT/IL-2, by which PSG-1 exerted its effects on the activation of spleen lymphocytes. Moreover, we further demonstrated that the PKC/NFAT/IL-2 signaling pathway cooperatively regulated PSG-1-induced spleen lymphocyte activation. However, because spleen lymphocytes consist of several subgroups, how PSG-1 affects a specific subgroup, such as T cell and B cell, is necessary to be clarified in further studies.

Figure 6. Effect of PKC inhibitor calphostin C on NFAT transactivation activity and IL-2 production induced by PSG-1 in spleen lymphocytes. (A) Calphostin C (25, 50, and 100 nM) was used to treat the transfected spleen lymphocytes for 30 min before PSG-1 treatment (160 μg/mL) for 48 h, and then luciferase activity was measured. (B) Cells were pre-incubated with calphostin C (25, 50, and 100 nM) for 30 min and then stimulated by PSG-1 (160 μg/mL) for 48 h. The supernatant was collected for measuring IL-2 production by ELISA. The data are expressed as the mean ± SEM for three separate experiments. (#) p < 0.05 versus the control group, and (∗) p < 0.05 versus the PSG-1 alone group.

NFAT, a family of four transcription factors first described in lymphocytes, has been found to express in almost all vertebrate cell types.32 To assess the effect of PSG-1 on NFAT activity in spleen lymphocytes, we performed transient transfection and detected the luciferase activity by the luciferase reporter assay. The data showed that PSG-1 was capable of elevating NFAT activity in a dose-dependent manner. Diverse transcription factors have been reported to be regulated by CaN in many cell types. Among these, NFAT is of special interest and best studied. In resting cells, NFAT resides in the cytoplasm in a phosphorylated form. Upon dephosphorylation by CaN, it subsequently translocates to the nucleus.33 In this study, we

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AUTHOR INFORMATION

Corresponding Authors

*Telephone/Fax: +86-791-88304452. E-mail: [email protected]. cn. E

DOI: 10.1021/acs.jafc.5b00028 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry *Telephone/Fax: +86-791-83969009. E-mail: [email protected]. cn.

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Funding

The financial support for this study by the National Key Technology Research and Development Program of China (2012BAD33B06), the Key Program of the National Natural Science Foundation of China (31130041), the Program for New Century Excellent Talents in University (NCET-120749), the Research Program of State Key Laboratory of Food Science and Technology (SKLF-ZZA-201301), the Project of Science and Technology of Jiangxi Provincial Education Department (KJLD13004), and the Key Project of International Cooperation of Jiangxi Provincial Department of Science and Technology (20141BDH80009) is gratefully acknowledged. Notes

The authors declare no competing financial interest.

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ABBREVIATIONS USED

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REFERENCES

CaN, calcineurin; CsA, cyclosporin A; IL-2, interleukin-2; NFAT, nuclear factor of activated T cells; PKC, protein kinase C

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