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Auraptene has the inhibitory property on murine T lymphocyte activation
European Journal of Pharmacology 750 (2015) 8–13
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
Auraptene has the inhibitory property on murine T lymphocyte activation Xinli Niu a, Zhihong Huang b, Lin Zhang c, Xuequn Ren c, Junpeng Wang c,d,n a
College of Life Science, Henan University, Kaifeng 475004, China College of Nursing, Henan University, Kaifeng 475004, China c Translational Medicine Center of Huaihe Hospital, Henan University, Kaifeng 475004, China d Institute of Molecular Medicine, Henan University, Kaifeng 475004,China b
art ic l e i nf o
a b s t r a c t
Article history: Received 8 July 2014 Received in revised form 13 January 2015 Accepted 15 January 2015 Available online 22 January 2015
Auraptene, a citrus fruit-derived coumarin, has been reported to exert valuable pharmacological properties as anti-tumor, anti-inflammatory, and anti-oxidant agent. However, little is known about auraptene on immune responses. In this study, we conducted an investigation to evaluate auraptene as an anti-T lymphocyte proliferation agent using CD3/CD28-activated lymphocytes isolated from C57BL/6 mice. We found that administration of auraptene inhibited CD3/CD28-activated lymphocyte proliferation in a dose dependent manner, but the inhibition at a wide range of doses used in this study did not induce cytotoxicity or apoptosis. In addition, auraptene dose dependently decreased the CD3/CD28activated T lymphocyte secreting T helper (Th)1 cytokines (interleukin (IL)-2 and interferon (IFN)-γ); whereas, auraptene could decrease Th2 cytokine (IL-4) at a higher level (40 mM) but had not at lower levels (10 and 20 mM). Further mechanistic study demonstrated that auraptene doses dependently suppressed T cell early and middle/late activation marker CD69 and CD25 expression, respectively. Finally, auraptene could suppress cell cycle progression which contributes to inhibiting T cell proliferation and cell division. These findings indicate that auraptene exhibits anti-inflammatory properties via inhibiting T cell proliferation and their inflammatory cytokine secretion that may mediate the interaction between T cells and autoimmune disorders, suggesting that auraptene is a potential food-derived compound with a benefit to those with abnormally over-activation T cell mediated response and chronic inflammation such as autoimmune and inflammatory diseases. & 2015 Elsevier B.V. All rights reserved.
Keywords: Auraptene Inflammation T cells Cytokines Cell cycle
1. Introduction Inflammation is the immune response of tissues due to bodily injury or infection. In an acute inflammation, it contributes to protecting and healing the body following physical injury or infection if it is highly regulated (Abdullah et al., 2011). However, if the stimulus persists, a chronic inflammation is mediated in early stage by monocytes and macrophages which persist in releasing the proinflammatory mediators, e.g., interleukin (IL)-6, IL-1β, tumor necrosis factors (TNF)-α, and prostaglandins, and in later stage by persistent activation of lymphocytes such as T and B lymphocytes which specifically target the invading microorganism or self-tissues for destruction. Several human diseases are inflammatory in nature, such as asthma, Crohn's disease, rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease (Gil, 2002). The etiology of several of n Corresponding author at: Translational Medicine Center of Huaihe Hospital, Henan University, Kaifeng 475004, China. Tel.: þ 86 150 93675738; fax: þ86 371 23906058. E-mail address: [email protected] (J. Wang).
http://dx.doi.org/10.1016/j.ejphar.2015.01.017 0014-2999/& 2015 Elsevier B.V. All rights reserved.
these diseases is still unknown; however, the lymphocyte overactivation, especially T cells, and their inflammatory cytokine secretion involve in the development of these diseases. Thus, regulating lymphocyte over-activation and reducing inflammatory mediators produced by lymphocytes may have the preventive or therapeutic potential to attenuate chronic inflammation mediated disorders. Epidemiological data suggest that dietary components from vegetables and fruits may modulate the inflammatory responses within body (Barbaresko et al., 2013; Holt et al., 2009). Polyphenols represent a major subset of phytochemicals and are widely distributed in vegetables and fruits (García-Lafuente et al., 2009). Studies have shown that dietary polyphenols reduce the risk of chronic diseases (Liu, 2004) and improve the autoimmune and transplantation (Abdullah et al., 2011; Hushmendy et al., 2009) via modifying the immune system (Middleton et al., 2000) and lowering the markers of inflammation (Holt et al., 2009). Auraptene is a citrus-derived polyphenol that belongs to the coumarin family (Borges et al., 2005; Genovese and Epifano, 2011). It is the most abundant prenyloxycoumarin and is mostly found in a variety of citrus fruits. Poncirus trifoliate, a traditional Chinese medicinal plant, contains a large
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quantity of auraptene in both peel and juice sac (Ogawa et al., 2000). Administration of auraptene has revealed to have medicinal and pharmacological properties including antioxidant, anti-bacterial, anti-inflammation, immunomodulation, and anti-cancer (Epifano et al., 2008; Kohno et al., 2006; Okuyama et al., 2014; Sekiguchi et al., 2012; Tanaka et al., 1999). However, little is known whether auraptene has the direct effect on T cell proliferation and activation. In the current investigation, to elucidate whether auraptene may directly modulate activated-lymphocyte induced inflammation and proliferation response, the effect of auraptene on the inflammatory cytokine production and T-lymphocyte proliferation responses were accessed using LPS- and CD3/CD28- activated lymphocytes, respectively. Next, the effect of auraptene on the production of T cell-secreted cytokines was analyzed. Finally, we addressed the mechanism of this effect on CD3/CD28-activated T lymphocytes.
2. Materials and methods 2.1. Mice Female C57BL/6 mice (6–8 wk) were purchased from the Animal Institute of Chinese Medical Academy (Beijing, China). Mice were housed three to four per cage in a controlled environment with 12:12 h light and dark cycle. They were provided free access to pathogen-free water and food. All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals by the National Institute of Health and approved by the Institutional Animal Care and Use Committee of Huaihe Hospital at Henan University. 2.2. Lymphocyte proliferation and division assay After mice were euthanized by cervical dislocation, inguinal lymph nodes were collected and a single cell suspension was prepared to perform lymphocyte proliferation and division. To determine lymphocyte proliferation, the lymphocytes (2 105 cells/well) in triplicate 96well, round-bottom culture plates were incubated with different concentrations of auraptene (AUR) (Sigma-Aldrich, St. Louis, MO) for 2 h and then stimulated with plated-coated anti-CD3 antibody (5 μg/ ml) and soluble anti-CD28 antibody (1 μg/ml) (CD3/CD28) (both from Biolegend, San Jose, CA) for 72 h. After that, MTT (Sigma) was added to each well and incubated for the final 4 h. The plates were centrifuged and the untransformed MTT was removed carefully by pipetting and then 200 μl of a DMSO (Sigma) working solutions was added to each well for the color development. Optical density (OD) was read at 570 nm by a plate reader. Stimulated index (SI) was calculated using the equation: SI¼ the mean OD of triplicate wells stimulated with CD3/CD28 in the absence or presence of AURCthe mean OD of triplicate wells stimulated with medium. To determine T cell division, the lymphocytes were stained using 0.5 μM Carboxyfluorescein succinimidyl ester (CFSE) as described previously (Tai et al., 2008). The CFSE-labeled cells at a density of 2 106/ml in a 24-well plate were incubated in the absence or presence of AUR for 2 h and then stimulated with CD3/CD28 for 72 h. After cells were stained with allophycocyanin (APC)-conjugated anti-CD3 antibody (Biolegend, San Diego, CA), CD3 þ T cell division was evaluated with FACSCalibur (BD Pharmingen, San Jose, CA) and data were analyzed using Flowjo 7.6 software (Tree Star Inc., Ashland, OR). 2.3. Cell viability assay The cell viability was accessed using MTT assay as described above. The lymphocytes were cultured in triplicate 96-well plates and incubated in the absence or presence of AUR for 72 h. At 4 h prior to culture termination, the MTT was added and the cells were
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continuously cultured until termination. DMSO was added to each well, and OD value was read at 570 nm by a plate reader. 2.4. Apoptosis analysis The lymphocytes were incubated at 2 106/well in a 24-well plate in the absence or presence of AUR for 2 h and then stimulated with CD3/CD28 for 72 h. Cells were stained with APCconjugated anti-CD3 antibody, fluorescein isothiocyanate (FITC)conjugated anti-Annexin V (Sigma), and Propidium iodide (PI) (Sigma) and then acquired with FACSCalibur. Data were analyzed using Flowjo 7.6 software. 2.5. Cell cycle analysis The lymphocytes at a density of 2 106/ml in a 24-well plate were incubated in the absence or presence of AUR for 2 h and then stimulated with CD3/CD28 for 48 h. At 45 min prior to culture termination, cells in culture were incorporated with BrdU (Sigma) to label newly synthesized DNA. After cells were surface-stained with APC-conjugated anti-CD3 antibody, they were fixed and permeabilized and then labeled with FITC-conjugated anti-BrdU antibody (Biolegend) and PI. Incorporated BrdU and total DNA levels were evaluated with FACSCalibur and data were analyzed using Flowjo 7.6 software. 2.6. Lymphocyte activation marker analysis The lymphocytes were incubated at 2 106/well in a 24-well plate in the absence or presence of AUR for 2 h and then stimulated with CD3/CD28 for 8 h for CD69 expression and for 24 h for CD25 expression. Cells were stained with APC-anti-CD3, phycoerythrin (PE)-anti-CD69, and PE-anti-CD25 antibodies (all from Biolegend) and then acquired with FACSCalibur. Data were analyzed using Flowjo 7.6 software. 2.7. Cytokine production After the lymphocytes were incubated at 2 106/well in a 24well plate in the absence or presence of AUR for 2 h and then stimulated with CD3/CD28 for 72 h, the supernatants were collected to determine the IL-2, IL-4, and IFN-γ levels using ELISA (all from BD Pharmingen). 2.8. Statistical analysis Results are expressed as means 7S.E.M. Statistical analyses were carried on using Prism 5.0 software. The significant difference was determined using one-way ANOVA followed by the Dunnett test. Value of Po 0.05 indicated statistical significance.
3. Results 3.1. Auraptene inhibits lymphocyte proliferation As shown in Fig. 1A, auraptene dose dependently suppressed CD3/CD28-activated lymphocyte proliferation but had no effect at a lower dose of 5 μM; whereas the inhibitory effects of auraptene at a wide range of doses may not be caused by its' cytotoxicity (Fig. 1B). Apoptosis plays many central roles in the development and survival of an organism. Thus, we next want to detect the lymphocyte apoptosis in the presence of auraptene. Our data showed that auraptene did not significantly induce apoptosis at a wide range of doses (Fig. 1C and D).
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Fig. 1. Auraptene does not affect lymphocyte viability and apoptosis but inhibits T cell proliferation. The lymphocytes isolated from C57BL/6 mice were incubated with AUR at indicated concentrations for 2 h and then stimulated by CD3/CD28 for 72 h for cell proliferation (A) and apoptosis (C) assays. To determine the effect of auraptene on lymphocyte viability, the lymphocytes were incubated with AUR for 48 h (B). Cell proliferation (A) and cell viability (B) was determined using MTT assay. To determine apoptosis, cells were stained with APC-conjugated anti-CD3, FITC conjugated anti-Annexin V, and PI before being analyzed using flow cytometer (C and D). Apoptotic cells are defined as Annexin V-positive and 7-AAD-negative cells. One experiment representative of 5 independent experiments is shown for analysis of apoptotic cells (C). Data are shown as mean7 S.E.M. of 5 independent experiments. *Po 0.05 and **Po 0.01 vs control (AUR, 0 mM). AUR, auraptene.
3.2. Effect of auraptene on the production of T cell cytokines by lymphocytes
3.4. The inhibitory effects of auraptene on T cell proliferation were caused by inhibition of cell division and cell cycle progression
The release of cytokines by lymphocytes plays a key role in many aspects of T cell function. Thus, we determined the effect of auraptene on the production of T cell cytokines. The results showed that addition of auraptene decreased the IL-2 and IFN-γ levels in a dose dependent manner (Fig. 2A and B); whereas, the IL-4 levels was reduced at a higher dose of auraptene (40 mM, Fig. 2C).
It is well known that multiple factors, e.g., antigen and mitogen signaling, costimulatory, and signaling from cytokines in resting T cells cooperate to induce cell division and cell cycle progression which contribute to driving T cell clonal expansion and differentiation. Thus, we determined whether auraptene inhibits lymphocyte clonal expansion by inhibiting T cell division and cell cycle progression. Compared to unstimulation, CD3/CD28-stimulation promoted T cell division (Fig. 4); whereas, auraptene dose dependently suppressed T cell division (Fig. 4). Since the reduced cell division may be due to the ability of cells to enter the different division phases, we next evaluated the inhibitory effect of auraptene on cell division by influencing cell cycle progression. As shown in Fig. 5, CD3/CD28-stimulation induced a significant decrease of the percent of G0/G1 phase compared to unstimulation. However, addition of auraptene resulted in an inhibition of G0/G1 phase, while the S phase cells showed a significant decrease (Fig. 5). These results indicate that auraptene can inhibit T cell proliferation partly via inducing G0/G1 phase cell cycle arrest of murine lymphocytes stimulated by CD3/CD28.
3.3. Auraptene suppresses T cell activation It is well established that lymphocyte activation is essential to induce inflammation. Thus, we determined whether the inhibitory effect of auraptene is via modulating T cell activation. We firstly measured the expression of CD69, the earliest activation marker, on activated T cells. The CD69 expression was at a low level on resting T cells but was rapidly up-regulated on CD3/CD28-activated T lymphocytes; whereas, the up-regulation of CD69 were inhibited by auraptene in a does dependent manner (Fig. 3A and B). IL-2 receptor α (CD25) is a middle or later activation marker on mitogen- or antigen-activated lymphocytes. Next we evaluated the CD25 expression on CD3/CD28-activated T lymphocytes. While CD3/ CD28-stimulation up-regulated the CD25 expression on CD3 þ T cells compared to unstimulation, the up-regulation of CD25 were reduced by auraptene at doses of 10–40 mM (Fig. 3C and D).
4. Discussion Lymphocytes (T and B cells), together with monocyte-derived macrophages, are common participants in inflammatory responses associated with various forms of bodily injury and infection (Ross,
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Fig. 2. Auraptene inhibits the production of T cell cytokines. After the lymphocytes isolated from C57BL/6 mice were incubated with AUR in the presence of CD3/CD28 for 72 h, the cell-free supernatants were collected to analyze the cytokine IL-2 (A), IFN-γ (B), and IL-4 (C) levels using ELISA. Data are shown as mean 7 S.E.M. of 5 independent experiments. *Po 0.05, **P o0.01, and ***P o0.001 vs control (AUR, 0 mM). AUR, auraptene.
Fig. 3. Auraptene suppresses the expression of CD69 and CD25 on T cells. After the lymphocytes isolated from C57BL/6 mice were incubated with AUR in the presence of CD3/CD28 for 8 h for CD69 expression and for 24 h for CD25 expression, the cells were collected to analyze the CD69 (A and B) and CD25 (C and D) expression on CD3 þ T lymphocytes. Data are from one experiment representative of 5 independent experiments (A and C). B and D: mean 7S.E.M. of five independent experiments (B and D). *Po 0.05, **P o 0.01, and ***P o 0.001 vs control with CD3/CD28 (AUR, 0 mM). AUR, auraptene.
1994). Previous studies have shown that auraptene, a citrus-derived component, is able to attenuate inflammatory responses in LPSactivated macrophage in vitro (Lin et al., 2013; Yan et al., 2013). In addition, an in vivo study has shown that auraptene may ameliorate LPS-induced inflammation in the mouse brain (Okuyama et al., 2014). In the present study, auraptene exerts its anti-inflammatory effects by inhibiting the production of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, and IL-12 from LPS-activated lymphocytes (Supplemental Fig. 1); whereas, these pro-inflammatory cytokines may initiate nonspecific inflammatory responses and induce T cell activation and differentiation. Thus, these data suggest that auraptene might have
the ability to influence T lymphocyte mediated immunities partly by inhibiting pro-inflammatory cytokine production from lymphocytes (mainly macrophage and B cells). However, it is unclear about the direct effect of auraptene on T cell proliferation and activation. Thus, we firstly evaluated the impact of auraptene on T cell proliferation using in vitro CD3/CD28-activated T cell model. Our data showed that auraptene supplementation dose dependently inhibits CD3 þ T cell proliferation but does not change the cell viability and apoptosis from CD3/CD28-activated lymphocytes, suggesting that this effect of auraptene is not through its toxicity to exert the property of anti- T cell proliferation (Fig. 1). However, which
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Fig. 4. Effect of auraptene on T cell division. After the lymphocytes isolated from C57BL/6 mice were stained with CFSE and incubated with AUR in the presence of anti-CD3/ CD28 antibodies for 72 h, the cells were collected to determine CD3 þ T lymphocyte division. One representative data of 5 is shown (A). B: mean 7 S.E.M. of 5 independent experiments. *P o 0.05, **Po 0.01, and ***P o0.001 vs control with CD3/CD28 (AUR, 0 mM). AUR, auraptene.
Fig. 5. Auraptene inhibits T cell cycle progression. After the lymphocytes isolated from C57BL/6 mice were incubated with 40 mM of AUR in the presence of CD3/CD28 for 48 h, cells were collected and co-stained with anti-BrdU and PI. Cell cycle was analyzed on CD3 þ T cells. Data are from one experiment representative of 5 independent experiments (A). B: mean 7 S.E.M. of five independent experiments. ***Po 0.001 vs control with CD3/CD28 (AUR, 0 mM). AUR, auraptene.
T cell subtype such as CD4 þ or/and CD8 þ T cell is inhibited by auraptene needs to be investigated. Quiescent T cell proliferation require two sequential mitogenic signals: the stimulation of the T-cell antigen receptor that triggers a complex activation program resulting in cell cycle entry from G0- into G1- phase (Firpo et al., 1994; Lea et al., 2003) and the expression of high affinity IL-2 receptor that subsequently binds to IL-2 to form IL-2/ IL-2 receptor complex eliciting the secondary signals to drive a G1- to S- phase transition, after which triggers T cell division (Nelson and Willerford, 1998). Based on the reduced IL-2 production and the down-regulated CD25 expression by auraptene, we speculate that auraptene has the ability to inhibit T cell division and cell progression. Indeed, we found that addition of auraptene reduced T cell division and prevented resting T cells from G0/G1 phase to S phase. In addition, the expression of CD69, the earliest T cell activation surface maker, was also reduced by auraptene when lymphocytes were stimulated by CD3/CD28. Thus, these data suggests that auraptene inhibits T cell
proliferation through impairing T cell activation and IL-2/IL-2 receptor signaling pathway. T cell-mediated immune response to antigen has been designated T helper (Th)1 and Th2 cell responses; whereas, Th1/Th2 cell balance plays a central role in modulating immune system and is polarized by inflammatory cytokine levels (Mosmann et al., 1986; O'Garra, 1998). Th1 cells, primarily secreting IL-2, TNF-α, and IFN-γ, are responsible for mediating cellular immunity to eradicate intracellular pathogens (Mosmann et al., 1986; Sher and Coffman, 1992). However, aberrant IFN-γ expression may also lead to immunopathology and organspecific autoimmune diseases (Hirahara et al., 2013; Liblau et al., 1995; O' Garra and Murphy, 1993). In vitro, naïve T cells can be differentiated to Th1 cells in the presence of IL-12 (Chang et al., 2000); whereas, auraptene inhibits the production of IL-12 by LPS-stimulated lymphocytes, indicating that auraptene might be able to reduce Th1 cell polarization by decreased IL-12 levels. Th2 cells, mainly secreting IL-4 and IL-13, are important to induce hormonal immunity to
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eliminate extracellular pathogens (Pulendran and Artis, 2012). However, Th2 secreting these various cytokines contribute to the development of allergy (Finkelman et al., 2010). Thus, attenuation of these cytokine levels (including Th1 and Th2 cytokine) might contribute to improving the development of autoimmune disorders. In the current study, addition of auraptene dose dependently inhibits Th1 cell secreting cytokines (IL-2 and IFN-γ) but only has this effect on the decreased IL-4 levels produced by Th2 cells at the highest dose. Previous study has shown (Tanaka et al., 1999) that oral administration of auraptene increased the IL-2 and IFN-γ production, not IL-4 production of splenocytes stimulated by ConA in mice; whereas, in the present study, we used an in vitro CD3/CD28-activated T cell model to find that addition of auraptene inhibits T cell secreting cytokines IL-2 and IFN-γ and has less effect on the IL-4 production. Therefore, we speculate that this different action of aurapetene on T cells might be due to the change of the coumarin structure after auraptene is oral or in vitro administrated.
5. Conclusion Taken all together, the result of this study showed that auraptene not only inhibits murine lymphocyte inflammatory responses but also impair primary T-cell proliferation. This T cell-suppressive action of auraptene is mediated via inhibiting cell division and cell cycle progression in T cells, the result of which may, in turn, be through impairing IL-2/IL-2 receptor signaling pathway. Further investigations are needed to directly address (1) how auraptene affects cell cyclerelated proteins in T cells and (2) whether and how auraptene affects Th cell differentiation. On the other hand, the anti-T cell proliferation property of auraptene may potentially be used as a remedy for autoimmune and inflammatory disorders.
Acknowledgments This work was supported in part by the National Natural Science Foundation of China (81402677), the Research Seed Fund (0000A40554), and the Initial Research Fund from Henan University (to J.W).
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Contents lists available at ScienceDirect
European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Immunopharmacology and inflammation
Auraptene has the inhibitory property on murine T lymphocyte activation Xinli Niu a, Zhihong Huang b, Lin Zhang c, Xuequn Ren c, Junpeng Wang c,d,n a
College of Life Science, Henan University, Kaifeng 475004, China College of Nursing, Henan University, Kaifeng 475004, China c Translational Medicine Center of Huaihe Hospital, Henan University, Kaifeng 475004, China d Institute of Molecular Medicine, Henan University, Kaifeng 475004,China b
art ic l e i nf o
a b s t r a c t
Article history: Received 8 July 2014 Received in revised form 13 January 2015 Accepted 15 January 2015 Available online 22 January 2015
Auraptene, a citrus fruit-derived coumarin, has been reported to exert valuable pharmacological properties as anti-tumor, anti-inflammatory, and anti-oxidant agent. However, little is known about auraptene on immune responses. In this study, we conducted an investigation to evaluate auraptene as an anti-T lymphocyte proliferation agent using CD3/CD28-activated lymphocytes isolated from C57BL/6 mice. We found that administration of auraptene inhibited CD3/CD28-activated lymphocyte proliferation in a dose dependent manner, but the inhibition at a wide range of doses used in this study did not induce cytotoxicity or apoptosis. In addition, auraptene dose dependently decreased the CD3/CD28activated T lymphocyte secreting T helper (Th)1 cytokines (interleukin (IL)-2 and interferon (IFN)-γ); whereas, auraptene could decrease Th2 cytokine (IL-4) at a higher level (40 mM) but had not at lower levels (10 and 20 mM). Further mechanistic study demonstrated that auraptene doses dependently suppressed T cell early and middle/late activation marker CD69 and CD25 expression, respectively. Finally, auraptene could suppress cell cycle progression which contributes to inhibiting T cell proliferation and cell division. These findings indicate that auraptene exhibits anti-inflammatory properties via inhibiting T cell proliferation and their inflammatory cytokine secretion that may mediate the interaction between T cells and autoimmune disorders, suggesting that auraptene is a potential food-derived compound with a benefit to those with abnormally over-activation T cell mediated response and chronic inflammation such as autoimmune and inflammatory diseases. & 2015 Elsevier B.V. All rights reserved.
Keywords: Auraptene Inflammation T cells Cytokines Cell cycle
1. Introduction Inflammation is the immune response of tissues due to bodily injury or infection. In an acute inflammation, it contributes to protecting and healing the body following physical injury or infection if it is highly regulated (Abdullah et al., 2011). However, if the stimulus persists, a chronic inflammation is mediated in early stage by monocytes and macrophages which persist in releasing the proinflammatory mediators, e.g., interleukin (IL)-6, IL-1β, tumor necrosis factors (TNF)-α, and prostaglandins, and in later stage by persistent activation of lymphocytes such as T and B lymphocytes which specifically target the invading microorganism or self-tissues for destruction. Several human diseases are inflammatory in nature, such as asthma, Crohn's disease, rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease (Gil, 2002). The etiology of several of n Corresponding author at: Translational Medicine Center of Huaihe Hospital, Henan University, Kaifeng 475004, China. Tel.: þ 86 150 93675738; fax: þ86 371 23906058. E-mail address: [email protected] (J. Wang).
http://dx.doi.org/10.1016/j.ejphar.2015.01.017 0014-2999/& 2015 Elsevier B.V. All rights reserved.
these diseases is still unknown; however, the lymphocyte overactivation, especially T cells, and their inflammatory cytokine secretion involve in the development of these diseases. Thus, regulating lymphocyte over-activation and reducing inflammatory mediators produced by lymphocytes may have the preventive or therapeutic potential to attenuate chronic inflammation mediated disorders. Epidemiological data suggest that dietary components from vegetables and fruits may modulate the inflammatory responses within body (Barbaresko et al., 2013; Holt et al., 2009). Polyphenols represent a major subset of phytochemicals and are widely distributed in vegetables and fruits (García-Lafuente et al., 2009). Studies have shown that dietary polyphenols reduce the risk of chronic diseases (Liu, 2004) and improve the autoimmune and transplantation (Abdullah et al., 2011; Hushmendy et al., 2009) via modifying the immune system (Middleton et al., 2000) and lowering the markers of inflammation (Holt et al., 2009). Auraptene is a citrus-derived polyphenol that belongs to the coumarin family (Borges et al., 2005; Genovese and Epifano, 2011). It is the most abundant prenyloxycoumarin and is mostly found in a variety of citrus fruits. Poncirus trifoliate, a traditional Chinese medicinal plant, contains a large
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quantity of auraptene in both peel and juice sac (Ogawa et al., 2000). Administration of auraptene has revealed to have medicinal and pharmacological properties including antioxidant, anti-bacterial, anti-inflammation, immunomodulation, and anti-cancer (Epifano et al., 2008; Kohno et al., 2006; Okuyama et al., 2014; Sekiguchi et al., 2012; Tanaka et al., 1999). However, little is known whether auraptene has the direct effect on T cell proliferation and activation. In the current investigation, to elucidate whether auraptene may directly modulate activated-lymphocyte induced inflammation and proliferation response, the effect of auraptene on the inflammatory cytokine production and T-lymphocyte proliferation responses were accessed using LPS- and CD3/CD28- activated lymphocytes, respectively. Next, the effect of auraptene on the production of T cell-secreted cytokines was analyzed. Finally, we addressed the mechanism of this effect on CD3/CD28-activated T lymphocytes.
2. Materials and methods 2.1. Mice Female C57BL/6 mice (6–8 wk) were purchased from the Animal Institute of Chinese Medical Academy (Beijing, China). Mice were housed three to four per cage in a controlled environment with 12:12 h light and dark cycle. They were provided free access to pathogen-free water and food. All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals by the National Institute of Health and approved by the Institutional Animal Care and Use Committee of Huaihe Hospital at Henan University. 2.2. Lymphocyte proliferation and division assay After mice were euthanized by cervical dislocation, inguinal lymph nodes were collected and a single cell suspension was prepared to perform lymphocyte proliferation and division. To determine lymphocyte proliferation, the lymphocytes (2 105 cells/well) in triplicate 96well, round-bottom culture plates were incubated with different concentrations of auraptene (AUR) (Sigma-Aldrich, St. Louis, MO) for 2 h and then stimulated with plated-coated anti-CD3 antibody (5 μg/ ml) and soluble anti-CD28 antibody (1 μg/ml) (CD3/CD28) (both from Biolegend, San Jose, CA) for 72 h. After that, MTT (Sigma) was added to each well and incubated for the final 4 h. The plates were centrifuged and the untransformed MTT was removed carefully by pipetting and then 200 μl of a DMSO (Sigma) working solutions was added to each well for the color development. Optical density (OD) was read at 570 nm by a plate reader. Stimulated index (SI) was calculated using the equation: SI¼ the mean OD of triplicate wells stimulated with CD3/CD28 in the absence or presence of AURCthe mean OD of triplicate wells stimulated with medium. To determine T cell division, the lymphocytes were stained using 0.5 μM Carboxyfluorescein succinimidyl ester (CFSE) as described previously (Tai et al., 2008). The CFSE-labeled cells at a density of 2 106/ml in a 24-well plate were incubated in the absence or presence of AUR for 2 h and then stimulated with CD3/CD28 for 72 h. After cells were stained with allophycocyanin (APC)-conjugated anti-CD3 antibody (Biolegend, San Diego, CA), CD3 þ T cell division was evaluated with FACSCalibur (BD Pharmingen, San Jose, CA) and data were analyzed using Flowjo 7.6 software (Tree Star Inc., Ashland, OR). 2.3. Cell viability assay The cell viability was accessed using MTT assay as described above. The lymphocytes were cultured in triplicate 96-well plates and incubated in the absence or presence of AUR for 72 h. At 4 h prior to culture termination, the MTT was added and the cells were
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continuously cultured until termination. DMSO was added to each well, and OD value was read at 570 nm by a plate reader. 2.4. Apoptosis analysis The lymphocytes were incubated at 2 106/well in a 24-well plate in the absence or presence of AUR for 2 h and then stimulated with CD3/CD28 for 72 h. Cells were stained with APCconjugated anti-CD3 antibody, fluorescein isothiocyanate (FITC)conjugated anti-Annexin V (Sigma), and Propidium iodide (PI) (Sigma) and then acquired with FACSCalibur. Data were analyzed using Flowjo 7.6 software. 2.5. Cell cycle analysis The lymphocytes at a density of 2 106/ml in a 24-well plate were incubated in the absence or presence of AUR for 2 h and then stimulated with CD3/CD28 for 48 h. At 45 min prior to culture termination, cells in culture were incorporated with BrdU (Sigma) to label newly synthesized DNA. After cells were surface-stained with APC-conjugated anti-CD3 antibody, they were fixed and permeabilized and then labeled with FITC-conjugated anti-BrdU antibody (Biolegend) and PI. Incorporated BrdU and total DNA levels were evaluated with FACSCalibur and data were analyzed using Flowjo 7.6 software. 2.6. Lymphocyte activation marker analysis The lymphocytes were incubated at 2 106/well in a 24-well plate in the absence or presence of AUR for 2 h and then stimulated with CD3/CD28 for 8 h for CD69 expression and for 24 h for CD25 expression. Cells were stained with APC-anti-CD3, phycoerythrin (PE)-anti-CD69, and PE-anti-CD25 antibodies (all from Biolegend) and then acquired with FACSCalibur. Data were analyzed using Flowjo 7.6 software. 2.7. Cytokine production After the lymphocytes were incubated at 2 106/well in a 24well plate in the absence or presence of AUR for 2 h and then stimulated with CD3/CD28 for 72 h, the supernatants were collected to determine the IL-2, IL-4, and IFN-γ levels using ELISA (all from BD Pharmingen). 2.8. Statistical analysis Results are expressed as means 7S.E.M. Statistical analyses were carried on using Prism 5.0 software. The significant difference was determined using one-way ANOVA followed by the Dunnett test. Value of Po 0.05 indicated statistical significance.
3. Results 3.1. Auraptene inhibits lymphocyte proliferation As shown in Fig. 1A, auraptene dose dependently suppressed CD3/CD28-activated lymphocyte proliferation but had no effect at a lower dose of 5 μM; whereas the inhibitory effects of auraptene at a wide range of doses may not be caused by its' cytotoxicity (Fig. 1B). Apoptosis plays many central roles in the development and survival of an organism. Thus, we next want to detect the lymphocyte apoptosis in the presence of auraptene. Our data showed that auraptene did not significantly induce apoptosis at a wide range of doses (Fig. 1C and D).
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Fig. 1. Auraptene does not affect lymphocyte viability and apoptosis but inhibits T cell proliferation. The lymphocytes isolated from C57BL/6 mice were incubated with AUR at indicated concentrations for 2 h and then stimulated by CD3/CD28 for 72 h for cell proliferation (A) and apoptosis (C) assays. To determine the effect of auraptene on lymphocyte viability, the lymphocytes were incubated with AUR for 48 h (B). Cell proliferation (A) and cell viability (B) was determined using MTT assay. To determine apoptosis, cells were stained with APC-conjugated anti-CD3, FITC conjugated anti-Annexin V, and PI before being analyzed using flow cytometer (C and D). Apoptotic cells are defined as Annexin V-positive and 7-AAD-negative cells. One experiment representative of 5 independent experiments is shown for analysis of apoptotic cells (C). Data are shown as mean7 S.E.M. of 5 independent experiments. *Po 0.05 and **Po 0.01 vs control (AUR, 0 mM). AUR, auraptene.
3.2. Effect of auraptene on the production of T cell cytokines by lymphocytes
3.4. The inhibitory effects of auraptene on T cell proliferation were caused by inhibition of cell division and cell cycle progression
The release of cytokines by lymphocytes plays a key role in many aspects of T cell function. Thus, we determined the effect of auraptene on the production of T cell cytokines. The results showed that addition of auraptene decreased the IL-2 and IFN-γ levels in a dose dependent manner (Fig. 2A and B); whereas, the IL-4 levels was reduced at a higher dose of auraptene (40 mM, Fig. 2C).
It is well known that multiple factors, e.g., antigen and mitogen signaling, costimulatory, and signaling from cytokines in resting T cells cooperate to induce cell division and cell cycle progression which contribute to driving T cell clonal expansion and differentiation. Thus, we determined whether auraptene inhibits lymphocyte clonal expansion by inhibiting T cell division and cell cycle progression. Compared to unstimulation, CD3/CD28-stimulation promoted T cell division (Fig. 4); whereas, auraptene dose dependently suppressed T cell division (Fig. 4). Since the reduced cell division may be due to the ability of cells to enter the different division phases, we next evaluated the inhibitory effect of auraptene on cell division by influencing cell cycle progression. As shown in Fig. 5, CD3/CD28-stimulation induced a significant decrease of the percent of G0/G1 phase compared to unstimulation. However, addition of auraptene resulted in an inhibition of G0/G1 phase, while the S phase cells showed a significant decrease (Fig. 5). These results indicate that auraptene can inhibit T cell proliferation partly via inducing G0/G1 phase cell cycle arrest of murine lymphocytes stimulated by CD3/CD28.
3.3. Auraptene suppresses T cell activation It is well established that lymphocyte activation is essential to induce inflammation. Thus, we determined whether the inhibitory effect of auraptene is via modulating T cell activation. We firstly measured the expression of CD69, the earliest activation marker, on activated T cells. The CD69 expression was at a low level on resting T cells but was rapidly up-regulated on CD3/CD28-activated T lymphocytes; whereas, the up-regulation of CD69 were inhibited by auraptene in a does dependent manner (Fig. 3A and B). IL-2 receptor α (CD25) is a middle or later activation marker on mitogen- or antigen-activated lymphocytes. Next we evaluated the CD25 expression on CD3/CD28-activated T lymphocytes. While CD3/ CD28-stimulation up-regulated the CD25 expression on CD3 þ T cells compared to unstimulation, the up-regulation of CD25 were reduced by auraptene at doses of 10–40 mM (Fig. 3C and D).
4. Discussion Lymphocytes (T and B cells), together with monocyte-derived macrophages, are common participants in inflammatory responses associated with various forms of bodily injury and infection (Ross,
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Fig. 2. Auraptene inhibits the production of T cell cytokines. After the lymphocytes isolated from C57BL/6 mice were incubated with AUR in the presence of CD3/CD28 for 72 h, the cell-free supernatants were collected to analyze the cytokine IL-2 (A), IFN-γ (B), and IL-4 (C) levels using ELISA. Data are shown as mean 7 S.E.M. of 5 independent experiments. *Po 0.05, **P o0.01, and ***P o0.001 vs control (AUR, 0 mM). AUR, auraptene.
Fig. 3. Auraptene suppresses the expression of CD69 and CD25 on T cells. After the lymphocytes isolated from C57BL/6 mice were incubated with AUR in the presence of CD3/CD28 for 8 h for CD69 expression and for 24 h for CD25 expression, the cells were collected to analyze the CD69 (A and B) and CD25 (C and D) expression on CD3 þ T lymphocytes. Data are from one experiment representative of 5 independent experiments (A and C). B and D: mean 7S.E.M. of five independent experiments (B and D). *Po 0.05, **P o 0.01, and ***P o 0.001 vs control with CD3/CD28 (AUR, 0 mM). AUR, auraptene.
1994). Previous studies have shown that auraptene, a citrus-derived component, is able to attenuate inflammatory responses in LPSactivated macrophage in vitro (Lin et al., 2013; Yan et al., 2013). In addition, an in vivo study has shown that auraptene may ameliorate LPS-induced inflammation in the mouse brain (Okuyama et al., 2014). In the present study, auraptene exerts its anti-inflammatory effects by inhibiting the production of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, and IL-12 from LPS-activated lymphocytes (Supplemental Fig. 1); whereas, these pro-inflammatory cytokines may initiate nonspecific inflammatory responses and induce T cell activation and differentiation. Thus, these data suggest that auraptene might have
the ability to influence T lymphocyte mediated immunities partly by inhibiting pro-inflammatory cytokine production from lymphocytes (mainly macrophage and B cells). However, it is unclear about the direct effect of auraptene on T cell proliferation and activation. Thus, we firstly evaluated the impact of auraptene on T cell proliferation using in vitro CD3/CD28-activated T cell model. Our data showed that auraptene supplementation dose dependently inhibits CD3 þ T cell proliferation but does not change the cell viability and apoptosis from CD3/CD28-activated lymphocytes, suggesting that this effect of auraptene is not through its toxicity to exert the property of anti- T cell proliferation (Fig. 1). However, which
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Fig. 4. Effect of auraptene on T cell division. After the lymphocytes isolated from C57BL/6 mice were stained with CFSE and incubated with AUR in the presence of anti-CD3/ CD28 antibodies for 72 h, the cells were collected to determine CD3 þ T lymphocyte division. One representative data of 5 is shown (A). B: mean 7 S.E.M. of 5 independent experiments. *P o 0.05, **Po 0.01, and ***P o0.001 vs control with CD3/CD28 (AUR, 0 mM). AUR, auraptene.
Fig. 5. Auraptene inhibits T cell cycle progression. After the lymphocytes isolated from C57BL/6 mice were incubated with 40 mM of AUR in the presence of CD3/CD28 for 48 h, cells were collected and co-stained with anti-BrdU and PI. Cell cycle was analyzed on CD3 þ T cells. Data are from one experiment representative of 5 independent experiments (A). B: mean 7 S.E.M. of five independent experiments. ***Po 0.001 vs control with CD3/CD28 (AUR, 0 mM). AUR, auraptene.
T cell subtype such as CD4 þ or/and CD8 þ T cell is inhibited by auraptene needs to be investigated. Quiescent T cell proliferation require two sequential mitogenic signals: the stimulation of the T-cell antigen receptor that triggers a complex activation program resulting in cell cycle entry from G0- into G1- phase (Firpo et al., 1994; Lea et al., 2003) and the expression of high affinity IL-2 receptor that subsequently binds to IL-2 to form IL-2/ IL-2 receptor complex eliciting the secondary signals to drive a G1- to S- phase transition, after which triggers T cell division (Nelson and Willerford, 1998). Based on the reduced IL-2 production and the down-regulated CD25 expression by auraptene, we speculate that auraptene has the ability to inhibit T cell division and cell progression. Indeed, we found that addition of auraptene reduced T cell division and prevented resting T cells from G0/G1 phase to S phase. In addition, the expression of CD69, the earliest T cell activation surface maker, was also reduced by auraptene when lymphocytes were stimulated by CD3/CD28. Thus, these data suggests that auraptene inhibits T cell
proliferation through impairing T cell activation and IL-2/IL-2 receptor signaling pathway. T cell-mediated immune response to antigen has been designated T helper (Th)1 and Th2 cell responses; whereas, Th1/Th2 cell balance plays a central role in modulating immune system and is polarized by inflammatory cytokine levels (Mosmann et al., 1986; O'Garra, 1998). Th1 cells, primarily secreting IL-2, TNF-α, and IFN-γ, are responsible for mediating cellular immunity to eradicate intracellular pathogens (Mosmann et al., 1986; Sher and Coffman, 1992). However, aberrant IFN-γ expression may also lead to immunopathology and organspecific autoimmune diseases (Hirahara et al., 2013; Liblau et al., 1995; O' Garra and Murphy, 1993). In vitro, naïve T cells can be differentiated to Th1 cells in the presence of IL-12 (Chang et al., 2000); whereas, auraptene inhibits the production of IL-12 by LPS-stimulated lymphocytes, indicating that auraptene might be able to reduce Th1 cell polarization by decreased IL-12 levels. Th2 cells, mainly secreting IL-4 and IL-13, are important to induce hormonal immunity to
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eliminate extracellular pathogens (Pulendran and Artis, 2012). However, Th2 secreting these various cytokines contribute to the development of allergy (Finkelman et al., 2010). Thus, attenuation of these cytokine levels (including Th1 and Th2 cytokine) might contribute to improving the development of autoimmune disorders. In the current study, addition of auraptene dose dependently inhibits Th1 cell secreting cytokines (IL-2 and IFN-γ) but only has this effect on the decreased IL-4 levels produced by Th2 cells at the highest dose. Previous study has shown (Tanaka et al., 1999) that oral administration of auraptene increased the IL-2 and IFN-γ production, not IL-4 production of splenocytes stimulated by ConA in mice; whereas, in the present study, we used an in vitro CD3/CD28-activated T cell model to find that addition of auraptene inhibits T cell secreting cytokines IL-2 and IFN-γ and has less effect on the IL-4 production. Therefore, we speculate that this different action of aurapetene on T cells might be due to the change of the coumarin structure after auraptene is oral or in vitro administrated.
5. Conclusion Taken all together, the result of this study showed that auraptene not only inhibits murine lymphocyte inflammatory responses but also impair primary T-cell proliferation. This T cell-suppressive action of auraptene is mediated via inhibiting cell division and cell cycle progression in T cells, the result of which may, in turn, be through impairing IL-2/IL-2 receptor signaling pathway. Further investigations are needed to directly address (1) how auraptene affects cell cyclerelated proteins in T cells and (2) whether and how auraptene affects Th cell differentiation. On the other hand, the anti-T cell proliferation property of auraptene may potentially be used as a remedy for autoimmune and inflammatory disorders.
Acknowledgments This work was supported in part by the National Natural Science Foundation of China (81402677), the Research Seed Fund (0000A40554), and the Initial Research Fund from Henan University (to J.W).
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