Glossogyne tenuifolia (Hsiang-ju) extract suppresses T cell activation by inhibiting activation of c-Jun N-terminal kinase

GT plant materials were purchased from an herb store in Penghu Island, Taiwan, and deposited as the number of ISU-JYH-001 in the Herbarium of I-Shou University (Kaohsiung City, Taiwan). The nucleotide sequences of internal transcribed spacer (ITS) 1, ITS2, and 5.8S rDNA were isolated from this plant, and the species was confirmed by the NCBI DNA database [4]. Dry whole plant materials were ground into powder. GT (5.3 kg) was extracted with 20 L of 95% ethanol at room temperature for 1 day and repeated for three times. The extracted solutions were filtered through medicinal gauze. The GTE was obtained by removing the solvent with a rotary evaporator (VP-60DJ, Panchum Co., Kaohsuing City, Taiwan) and drying in a freeze drier (FD8530, Panchum Co.). The total dry weight of this extract was 777 g, and the extraction yield was 14.7%. The quality of the herb extract was monitored by high performance liquid chromatography (L-7100, Hitachi, Tokyo, Japan) analysis using luteolin and luteolin-7-glucoside (Sigma Chemicals Co., St Louis, MO, USA) as the principle control standards [4, 9]. The dry GTE was dissolved in dimethyl sulfoxide (DMSO) at the concentration 50 mg/mL as the GTE stock. The final GTE concentrations (3.13, 6.3, 12.5, 25 and 50 μg/mL) were made by culture medium dilution. The DMSO was added no more than 0.1% (v/v) and used as a mock control in this study.

Human PBMCs were prepared from the whole blood and human leukemia cell line Jurkat was obtained from American Type Cell Collection (ATCC, Manassas, VA, USA). Human PBMCs and leukemic Jurkat cells were grown in RPMI-1640 medium (GE Healthcare, Piscataway, NJ, USA) with 10% fetal bovine serum (FBS; Thermo Fisher Scientific Inc., Waltham, MA, USA), 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and 2 mM l-glutamine (Sigma-Aldrich) at 37 °C in a humidified atmosphere with 5% CO₂. Human PBMCs were isolated from whole blood using a Ficoll-Paque plus density gradient [25]. Whole bloods from healthy volunteers were mixed with the same volume of Hank’s balanced salt solution (HBSS; Thermo Fisher, St. Louis, MO, USA). The blood-HBSS mixture was layered over Ficoll-Paque PLUS (GE Healthcare) 1:1 (v/v) and centrifuged (5810R, Eppendorf, Hamburg, Germany) (400×g) for 20 min at 25 °C with the break off. The PBMC layer at the interface was collected and washed with HBSS three times by centrifugation (300×g) for 5 min at 4 °C. The PBMCs were suspended in RPMI-1640 medium. The study protocols were approved by the Institutional Review Board of the Research Ethics Committee of National Health Research Institutes (EC1001101) (Additional file 1).

For each sample, 1 × 10⁶ cells were washed with phosphate buffered saline (PBS; pH 7.2) and lysed in freshly prepared cell lysis buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 µg/mL leupeptin, and 1 mM phenylmethanesulfonyl fluoride). The cell lysate was separated from debris by centrifugation at 13,200×g for 10 min at 4 °C. The protein concentration was measured by Bio-Rad Protein Assay Dye Reagent (Bio-Rad Laboratories, Hercules, CA, USA). An aliquot of 20 μg protein was separated on a 10% polyacrylamide gel and transferred onto a polyvinylidene fluoride membrane (EMD Millipore, Billerica, MA, USA). The membranes were subsequently blocked with 5% dry skim milk in Tris-buffered saline (TBS; pH 7.5). The membranes were hybridized with the indicated primary antibody at 4 °C overnight, washed three times with 0.05% TBST solution (50 mM Tris–Cl, pH 7.5, 150 mM NaCl, and 0.05% Tween-20) for 10 min, and then hybridized with a horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. After washing three times with 0.05% TBST for 10 min each, the membrane was incubated with Luminata™ Western HRP Substrates (EMD Millipore). The signals were detected using Image Lab software Version 2.0 (Bio-Rad Laboratories).

PBMCs (1 × 10⁵) were pretreated with GTE (3.13, 6.3, 12.5, 25 and 50 μg/mL) for 30 min and then activated by 2.5 μg/mL phytohemagglutinin (PHA) in each well of a 96-well plate for 48 h. An aliquot of 0.5 μCi ³H-thymidine was added to each well. After 20 h of incubation, the cells were harvested with a FilterMate 96-well harvester (PerkinElmer Inc, Waltham, MA, USA) for a proliferation assay. The radioactivity (counts per minute, CPM) was measured to quantify cell proliferation with a microplate scintillation and luminescence counter (TopCount NXT, Packard Instrument Co, Meriden, CT, USA). Jurkat cells (1 × 10⁵) were pretreated with GTE (25 μg/mL) for 30 min and then stimulated by 10/80 ng/mL 12-O-tetradodecanoyl-phorbol-13-acetate (PMA)/ionomycin (P/I) in each well of a 96-well plate for 24 h. Cells were then harvested for surface marker staining. To detect NF-κB activation, phosphorylation of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor (IκB) and p65 proteins were determined by western blotting. Jurkat cells (1 × 10⁶) were pretreated with GTE (25 μg/mL) for 30 min and then stimulated by PHA (2.5 μg/mL) for 0, 1, and 3 h. Cell lysates were then prepared for western blot analysis. To detect MAPK activation (p-ERK, p-p38, and p-JNK), 1 × 10⁶ Jurkat cells were pretreated with GTE (25 μg/mL) for 30 min and then stimulated with 2.5 μg/mL PHA for 0, 10 and 20 min. Cell lysates were then prepared for western blotting. Primary antibodies against p-IκB (#2859, 1:500), p-p65 (#3033, 1:2000), p-ERK (#9101, 1:3000), p-p38 (#4511, 1:1000), and p-JNK (#4671, 1:500) were purchased from Cell Signaling Technology (Beverly, MA, USA). Primary antibodies against α-tubulin (#2871, 1:3000) was purchased from Epitomics Inc. (Burlingame, CA, USA). Primary antibodies against ERK2 (sc-154, 1:5000), p38 (sc-535, 1:3000), and JNK1 (sc-1648, 1:1000), and an HRP-conjugated secondary antibody were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Jurkat cells (1 × 10⁵) were stimulated with P/I (10/80 ng/mL) for 24 h. The cells were then stained with CD69-PE (12-0699, 1:50, eBioscience, San Diego, CA, USA) in staining buffer (PBS with 1% FBS) on ice for 20 min and then washed three times with staining buffer. The CD69-positive population was evaluated by a flow cytometer (FACSCalibur; BD Biosciences). Data were analyzed with FlowJo software (BD Biosciences). The signal obtained for the cells treated without or with GTE alone and stained with mouse IgG1 isotype control-PE (12-4714, 1:50, eBioscience) was used as a control respectively.

Apoptosis was determined by propidium iodide (PI) (Sigma-Aldrich) staining [26]. PBMCs or Jurkat cells (1 × 10⁵) were treated with various concentrations of GTE (3.13, 6.3, 12.5, 25 and 50 μg/mL) in each well of a 96-well plate for 24 h. Harvested cells were suspended in a hypotonic solution (0.1% sodium citrate, 0.1% Triton X-100, and 20 μg/mL PI). The extent of the sub-G0/G1 peak representing apoptotic cells was measured by gating on cells stained in the region below the G0/G1 peak. The apoptotic sub-G0/G1 population was examined by flow cytometry and analyzed with FlowJo software (BD Biosciences).

Healthy female Balb/c mice were obtained from National Laboratory Animal Center (NLAC, Taipei, Taiwan) at 4 weeks of age. The animals were maintained in cages (5 mice/cage) under standard conditions. The mice were euthanized by use of CO₂ exposure. CD4⁺ T cells isolated from the splenocytes of C57BL/6 mice were activated by antibodies against CD3 (16-0031, eBioscience) and CD28 (16-0281, eBioscience) (1 and 2 μg/mL, respectively) in the presence of 100 U/mL IL-2 for 5 days. The activated T cells were washed three times and reactivated by the anti-CD3 (16-0031, eBioscience) antibody (0.5 μg/mL) for 8 h. The cells were then harvested for an apoptosis assay. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of I-Shou University (IACUC-ISU-96002) (Additional file 2). The combination of P/I and sodium orthovanadate (Na₃VO₄) induces significant AICD in Jurkat T cells [27]. Jurkat cells (1 × 10⁵) were pretreated with GTE (25 μg/mL) for 30 min and then activated by P/I (10/80 ng/mL) and 1.5 mM Na₃VO₄ in each well of a 96-well plate for 24 h. The cells were then harvested for an apoptosis assay.

Each experiment was performed at least three times and measured in triplicates. The data were presented as mean ± standard deviation (SD). Each GTE-treated group was compared to the respective mock-treated (DMSO) control group. Statistical differences were analyzed by Student’s t-test (*P < 0.05, **P < 0.01 and ***P < 0.001). Data were analyzed using GraphPad Prism software (GraphPad Software, La Jolla, CA, USA).

Details of our experimental design and statistics and all resources used in this study were included in Additional file 3.

Mitogen stimulation hydrolyzes membrane polyphosphoinositides to inositol trisphosphate (IP3) and diacylglycerol (DAG). Then, IP3 releases Ca²⁺ into the cytosol, and DAG activates protein kinase C (PKC). These synergistic effects lead to progression of the cell cycle and proliferation of T cells [28, 29]. A combination of phorbol esters, which activates PKC, and a calcium ionophore, which increases the intracellular Ca²⁺ concentration, mimic the signals triggered by the TCR or mitogen stimulation and can be used as agents for T cell activation. In addition, CD69 is an early activated surface molecule on T cells, and expression of CD69 has been frequently used as an indicator for T cell activation [30].

GTE has been reported to exert an inhibitory effect on LPS-treated macrophage and human PBMC activation [10, 11]. To further explore the influence of GTE on adaptive immunity, we examined the effect of GTE on the activation of T cells. PHA (a mitogen) or a combination of PMA (a phorbol ester) and ionomycin (a calcium ionophore) were used to activate T cells. First, Jurkat cells were pretreated with 25 μg/mL GTE for 30 min and then P/I were added to induce T cell activation for 24 h. The expression of CD69 was used as the T cell activation marker [31]. Figure 1a shows that GTE treatment reduced the fluorescence intensity of CD69-PE on P/I-activated Jurkat cells. The mean fluorescence intensity (MFI) was determined after analyzing 10,000 cells. The MFI changes of GTE and mock control were expressed as the fold changes relative to the individual group without P/I stimulation. The relative MFI fold changes of GTE-treated and mock control were 2.69 ± 0.01 and 4.14 ± 0.12 respectively. These data demonstrated that GTE administration decreased P/I-induced CD69 expression on P/I-activated Jurkat cells (P = 0.0044). In addition, the proliferation of primary T cells, representing successful activation of T cells, was used to evaluate T cell activation of PHA-stimulated PBMCs. Proliferation of PBMCs triggered with PHA (2.5 μg/mL) was suppressed by GTE/PHA cotreatment compared with PHA treatment alone at the GTE concentrations of 12.5, 25 and 50 μg/mL (P = 0.0118, 0.0030 and 0.0021) (Fig. 1b). The results of ³H-thymidine incorporation of PHA-activated PBMCs from four individual donors showed the suppressive trend of GTE in a dose-dependent manner (Additional file 4). The inhibitory effect of GTE on CD69 expression of activated Jurkat T cells (Fig. 1a) correlated well with the proliferation of PHA-stimulated human PBMCs in which GTE inhibited PHA-induced proliferation by up to 80% at 25 μg/mL (Fig. 1b). These results indicated that the GTE inhibited activation of both Jurkat T cells and human PBMCs.

Cytotoxic activity of GTE has been reported in certain cancer cell lines such as HepG2, Hep3B, MDA-MB-231, and MCF-7 [4, 7, 32]. This study showed that the death of cancer cells was mediated through the apoptotic pathway. The downregulating effect of GTE on T cell activation could be due to a cytotoxic effect on T cells. Thus, to determine whether GTE has a cytotoxic effect on T cells, Jurkat cells were cultured in the presence of various doses of GTE for 24 h, and then the degree of apoptosis was examined. Figure 2a shows minimal apoptosis in Jurkat cells (<20%) treated with GTE at up to 50 μg/mL. In addition, almost no apoptosis was observed in PBMCs treated with GTE at less than 25 μg/mL (Fig. 2b). Because GTE did not induce apoptosis of human PBMCs at 25 μg/mL, this dosage was used for the following activation assay. These results suggested that the inhibitory effect of GTE on T cell activation was not due to a cytotoxic effect.

The T cell activation signal activates multiple downstream molecules including phosphorylation of MAPKs and NF-κB. It has been shown that inhibition of the DNA-binding activity of NF-κB upon PHA stimulation is significant at a high dosage of GTE (up to 150 μg/mL) but insignificant at a low GTE dose (less than 100 μg/mL) [12]. In this study, we further examined GTE effects on NF-κB activity in T cell activation signaling. In the NF-κB activation pathway, IκB is phosphorylated and undergoes degradation upon PHA stimulation. We therefore evaluated IκB phosphorylation upon T cell activation. Figure 3 shows that GTE-treated Jurkat cells displayed similar levels of IκB phosphorylation upon PHA stimulation. The phosphorylation of RelA/p65, one of the NF-κB members, regulates NF-κB transcriptional activity [33]. We found that RelA/p65 phosphorylation was normal after co-treatment with GTE (Fig. 3). These data suggest that the deficiency of T cell activation in the presence of GTE is independent of the NF-κB pathway.

Because NF-κB signaling was normal in GTE-treated Jurkat cells, we mapped the signal defect in these cells. In addition to NF-κB signaling, the activities of MAPKs are important for T cell activation. Thus, we further examined the activity of three major MAPKs, ERK, p38 MAPK, and JNK, after stimulation. T cell activation increased ERK phosphorylation, and GTE administration did not alter the phosphorylation status of ERK (Fig. 4a, b). GTE slightly inhibited p38 MAPK phosphorylation after stimulation with PHA for 20 min (P = 0.0278) (Fig. 4a, c). Notably, JNK phosphorylation was significantly suppressed by GTE in activated Jurkat cells at the activation time 10 and 20 min (P = 0.0217 and 0.0167) (Fig. 4a, d). Taken together, the suppression of T cell activation by GTE might be attributed to a slight reduction in p38 activation and profound JNK activation.

JNK is activated by a wide array of stress conditions and mitogenic stimulation. Previous studies have shown that JNK plays an important role in AICD [27]. It is important to examine whether GTE regulates AICD by modulating the activity of JNK. Na₃VO₄, a tyrosine phosphatase inhibitor, has been used to prolong JNK activation during T cell activation, whereas the combination of P/I and Na₃VO₄ upon T cell stimulation induces significant AICD in Jurkat T cells [27]. In this study, Na₃VO₄ was also shown to induce apoptosis upon T cell activation by P/I. Furthermore, administration of GTE with Na₃VO₄ or P/I did not induce apoptosis of Jurkat T cells (Fig. 5a). Figure 5a also demonstrates that GTE blocked Na₃VO₄-induced apoptosis in activated Jurkat T cells (P = 0.0037). Next, we examined the effect of GTE on AICD in reactivated T cells ex vivo. Naïve CD4 T cells were activated with anti-CD3/CD28 antibodies and maintained in IL2-containing medium for 5 days. The activated CD4 T cells were then re-activated with the anti-CD3 antibody to trigger AICD. Figure 5b shows that T cells underwent AICD through repeated stimulation of CD3, and GTE inhibited AICD of primary isolated T cells (P = 0.0016). These results suggested that GTE might attenuate prolonged JNK-induced AICD.