Numerous phytochemicals produced by plants as secondary metabolites are potent immunomodulators and their biological activity was demonstrated
Activation of T lymphocytes during an immune response is mediated by various T-lymphocyte receptors and triggers a series of programmed gene regulations, proliferation, and effector functions. Concanavalin A (CoA), a lectin isolated from the seeds of
In this study, we aimed to investigate the concentration-dependent effect of SIL on proliferation of CoA-stimulated splenocytes, viability of cells, and secretion of IFN-g and IL-4 cytokines
Spleen cells were incubated in RPMI medium (Biochrom-Merck, Germany) containing 2 mM of stable glutamine and supplemented with 10% heat-inactivated bovine fetal serum (Biochrom-Merck, Germany), 100 U/ml penicillin, 100 μg/ml streptomycin, 10 μg/ml gentamicin, and 2.5 μg/ml amphotericin B (all from Sigma-Aldrich, St. Louis, USA). SIL was purchased from Sigma-Aldrich and was dissolved in 100% DMSO to obtain a concentration of 10 mg/ml. All the subsequent dilutions were made in RPMI medium and the final DMSO concentration was 0.1% or lower. Cells were treated with SIL concentrations of 2.5, 5, 10, 20, and 40 μg/ml (final). The composition of SIL was determined by HPLC analysis (Hrčková et al., in press) and was as follows: taxifolin (4%), silychristin A/silychristin B and silydianin (33%), silybin A/B (52%), isosilybin A/B (11%). CoA, Rhodamine 123, and DMSO were purchased from Sigma-Aldrich.
Male Balb/c mice were purchased from VELAZ (Prague, Czech Republic) and used at the age of 8 weeks. Spleens were aseptically isolated from three mice in each experiment (
The suspensions of naive splenocytes were diluted to a concentration of 1×106 cells/ml and seeded into flat bottom 96-well plates in quadruplicates (Corning) for each treatment. They were used to examine the effect of SIL on the T-lymphocyte proliferation by BrdU Cell Proliferation ELISA Kit (Roche Diagnostics GmbH, Mannheim, Germany) and cytokine production
Supernatants from other CoA-stimulated splenocyte cultures (not supplemented with BrdU) were collected. Samples from identical treatments in wells were pooled and stored at −80°C for determination of IFN-γ and IL-4 by ELISA kits (Mouse Ready-SET-Go ELISA; eBioscience, Germany). Then cytokine concentrations were determined in triplicates for each treatment/mouse using MaxiSorp Nunc-immuno module strips (Thermo Fisher Scientific, Roskilde, Denmark) and calculated in picogram per milliliter and expressed as mean ± SD.
Viability of CoA-stimulated splenocytes was evaluated after 70 h of incubation with increasing SIL concentrations using 0.05% solution of trypan blue (Sigma-Aldrich, Merck Sigma, UK) in PBS. Splenocytes were incubated in 24-well plates at a concentration of 1 × 106/ml in RPMI medium, stimulated with CoA, and treated with the same concentrations of SIL (in quadruplicates) as were used in the proliferation assay. After 70 h of incubation, nonadherent cells (lymphocytes) from each well were collected in tubes, and after addition of trypan blue, unstained live cells were counted using hemocytometer. Proportions (%) of live cells from total counted cells per well (
Naive, unstimulated, and CoA-stimulated cells treated with SIL were incubated in 24-well plates for 70 h as described previously. Then nonadherent cells were collected into tubes and used to measure mitochondrial membrane potential (Ym) using the fluorescent dye Rhodamine 123, which is taken by mitochondria of living cells. Changes in the uptake of this dye are believed to reflect the level of Ym (Darzynkiewicz et al., 1982). The stock solution (1 mg/ml) of Rhodamine 123 was prepared in ethanol and added to the cell suspensions. Cells were incubated with Rhodamine 123 (10 μM final) for 20 min at 37°C and immediately used to measure Ym without washing by flow cytometry. Flow cytometry was performed with excitation of Rhodamine 123 at 505 nm and emission at 535 nm using flow cytometer FACS Canto (Becton Dickinson Biosciences, USA). Data were analyzed using FACS Diva software and were expressed as the mean fluorescence intensity of Rhodamine 123 to establish the effect of CoA and SIL on splenocytes.
Cell suspensions of nonadherent cells from cultivations in 24-well plates treated in the same way as were applied in other assays were used to study apoptotic process after 70 h of incubation. Apoptosis was detected with BD Pharmingen Annexin V-FITC Apoptosis Detection Kit (APO Alert Annexin V, ClonTech, California, USA) according to manufacturer's instructions. Briefly, treated cells were centrifuged for 10 min to remove the medium, then were washed and resuspended in 200 μl of the binding buffer. Apoptotic cells were detected after staining with 5 μl of Annexin V and 5 μl of propidium iodide solutions at room temperature in the dark for 15 min. Analysis was performed by flow cytometry using BD FACS Canto (Becton Dickinson Biosciences) and data were analyzed using FACS Diva software. Data are expressed as proportions of apoptotic cells (%).
Quantitative transcription profiles of genes for IFN-g, IL-4, Foxp3, and NF-kB and housekeeping genes β-actin as well as GAPDH in spleen cells were determined by real-time PCR (RT-PCR). Cells (1×106/ml) were plated into 24-well plates in triplicate for each treatment and stimulated with CoA and/or SIL at increasing concentrations for 70 h. Then supernatants containing lymphocytes were collected into tubes, centrifuged, and pellets from three wells/treatment were pooled and used for extraction of RNA after adding Trizol reagent (Amresco, Solon, OH, USA). Then 3 μg of total RNA was reverse transcribed with RevertAid H Minus M-MuLV Reverse Transcriptase and oligo dT primers (both from Thermo Fisher Scientific, St. Leon-Rot, Germany). Real-time quantitative analysis of the relative abundance of mRNA species was determined using the SYBR green master mix (BioRad, Hercules, CA, USA) on BioRad CFX thermocycler (BioRad). PCR was performed in 20-μl reactions with detection primer pairs for IFN-γ (forward: 5′-TCAAGTGGCATAGATGTGGAAGAA-3′; reverse: 5′-TGGCTCTGCAGGATTTTCATG-3′), IL-4 (forward: 5′-ACAGGAGAAGGGACGCCAT-3′; reverse: 5′-GAAGCCCTACAGACGAGCTCA-3′), Foxp3 (forward: 5′-AAT AGT TCC TTC CCA GAG-3′; reverse: 5′-GAT TTC ATT GAG TGT CCT-3′), NF-kB (forward: 5′-GGGCAGTGACGCGACG-3′; reverse: 5′-AGCGCCCCTCGCATTTATAG-3′), β-actin (forward: 5′-ACCAACTGGGACGACATGGAGAAAATC-3′; reverse: 5′-GTAGCCGCGCTCGGTGAGGATCTTCAT-3′), and GAPDH (forward: 5′-TCACCACCATGGAGAAGGC-3′; reverse: 5′-GCTAAGCAGTTGGTGGTGCA-3′). The results are expressed as fold amplification of the target gene compared with its expression in cells in naive mice by means of the comparative Ct method DDCt (Livak and Schmittgen, 2001).
The results were analyzed either by using one-way ANOVA followed by Tukey's post hoc test or with the grouped analyses utilizing two-way ANOVA and the Sidak post hoc test. Data were evaluated by GraphPad Prism (version 7) (GraphPad Software, Inc., San Diego, CA, USA), and the differences were regarded as significant at least at p< 0.05.
CoA is a strong mitogen, activating proliferation preferentially of T-cell subsets via cell membrane receptors. Splenocytes were treated with increasing concentrations of SIL and PI was determined in BrdU incorporation assay. As shown in Fig. 1a, SIL stimulated proliferation up to a concentration of 5 μg/ml and significant elevation was detected in CoA-alone treated cells (
To investigate whether highly reduced proliferation is due to the cytotoxic effect of SIL on lymphocytes, we counted viable cells after staining with trypan blue and calculated the proportions of live cells from total counted cells (Fig. 1b). In comparison with unstimulated lymphocytes, the proportions of live cells increased significantly after CoA treatment and after CoA + 5 μg/ml of SIL exposure, which correlated with the changes of PI. However, at 40 μg/ml of SIL no cytotoxic effect was determined as non-significantly reduced numbers of live cells were observed, indicating that reduction of proliferation by SIL was caused by other mechanisms.
The effect of treatments on IFN-g and IL-4 cytokine levels and mRNA expression of both genes were examined by ELISA test and quantitative RT-PCR, respectively. As shown in Fig.2 a, b concentration of cytokines was barely detectable in unstimulated cells, but has markedly increased after CoA stimulation. SIL significantly enhanced production of IFN-g at concentrations of 5 and 10 μg/ml in comparison with CoA-alone-stimulated cells, but had no inhibitory effect at the highest concentration. In contrast, IL-4 levels were not significantly changed, except for the markedly suppressed secretion after exposure to 40 μg/ml of SIL. However, SIL treatment had the opposite effect on mRNA transcript levels for both cytokines (Fig.2 c, d). In unstimulated cells, the abundance of mRNA copies was several times higher than in CoA-stimulated cells. We found stimulation of genes expression with increasing concentrations of SIL in T-lymphocyte cell cultures, which was significantly higher (
SIL can suppress nuclear transcription factor NF-κB on the protein level but it is not clear if SIL similarly suppresses NF-kB on the mRNA level. To clarify concentration-dependent effect of SIL on CoA-stimulated T cells we examined the relative transcripts abundance of gene for p65 (RelA) which is one of the two subunits of NF-kB (Fig.3a). No difference was found between unstimulated and CoA-stimulated cells. Interestingly, SIL in dose-dependent manner stimulated expression of gene for this protein, and significantly elevated mRNA copies were found at a dose of 40 μg/ml (
Upon activation, T cells utilize mitochondrial energy to differentiate into distinct T-helper cells (Ozay et al., 2018) and changes in mitochondrial dynamic can be assessed by mitochondrial membrane potential (Ym). The effects of SIL on Ym in CoA-stimulated T lymphocytes are demonstrated in Fig. 4 as the mean intensity of fluorescence (MIF), and representative plots of MIF are shown in the left panel. In comparison with freshly isolated lymphocytes, incubation of naïve unstimulated cells for 70 h at standard conditions resulted in significant decrease of Ym (
Impairment in mitochondrial biogenesis to a critical level might result in cell death in many pathological conditions. To get a deeper insight into the regulation of T-cell functions and fate by various concentration of SIL, we determined the proportions of live cells, cells in the early stage of apoptosis, and dead cells by flow cytometry. Results of our analyses are summarized in Fig.5 and representative dot plots from analysis of freshly isolated naïve spleen cells (upper left panel) and CoA-stimulated cells treated with 5 μg/ml of SIL for 70 h are on lower left panel. While in suspension of freshly isolated spleen cells, more than 93% of cells were live, cultivation for 70 h negatively influenced the physiological state of lymphocytes as manifested by their entry into the early phase of apoptosis. We found that 86.2 ± 2.7% of unstimulated cells (control group) was at the early stage of apoptosis at the end of cultivation. Following CoA stimulation the proportion of early apoptotic cells decreased, whereas the proportions of live cells significantly increased (
SIL is a natural product widely studied for its numerous beneficial effects on health and prevention of many diseases (Gažák et al., 2007). It is considered very safe as after oral administration the 50% lethal dose was 10 g/kg in rats (Fraschini et al., 2002). Studies on immunomodulatory activities indicate concentration-dependent effect at various
Reports on concentration-dependent effect of SIL administration to mice present various findings. Johnson et al. (2003) showed that the absolute numbers of splenic CD3+ T lymphocytes were reduced after intraperitoneal administration of 10 and 50 mg/kg of SIL; however, PHA-induced T-lymphocyte proliferation
We further investigated the effect on cytokine secretion by CoA-stimulated cells and showed that only the highest concentration of SIL (40 μg/ml) leveled down the amount of IFN-γ and IL-4, probably due to diminished cell numbers as a consequence of reduced proliferation. The higher susceptibility to SIL was found for CD4+ T-helper cells activated with monoclonal anti-CD3/anti-CD28 antibodies, where SIL at concentrations of 20 and 30 μg/ml significantly decreased IFN-γ gene expression and upregulated Foxp3 transcription factor in Treg cells. Foxp3 factor plays a key role in the maintenance of lymphoid homeostasis in a number of immune circumstances, where Tregs guide immunosuppressive reactions (Feuerer et al., 2009).
In our study, SIL at the dose of 20 and 40 μg/ml stimulated expression of Foxp3 transcription factor in CoA-stimulated T lymphocytes, as well as mRNA levels for IFN-γ cytokine, suggesting that CoA probably induced a heterogeneous population of T cells with different transcriptional profiles and cytokine secretion. Stimulation of gene for NF-κB p65 subunit on mRNA level with increasing concentrations of SIL was also observed in our study. In the cytoplasm, NF-κB dimer is present in inactive form bound to inhibitory kappa B (IκB) protein and upon activation of cells the NF-κB is released from IκB and translocates to the nucleus. It was shown that SIL can block the translocation of NF-κB to nucleus via suppression of IκB degradation (Manna et al., 1999). We assume that although mRNA and probably also protein levels of NF-κB subunit were significantly elevated after 40 μg of SIL, simultaneous dose-dependent inhibition of IκB degradation occurred with increasing concentrations of SIL.
Interaction of CoA (concentration 3 μg/ml) with cell surface glycoproteins can induce apoptosis in fibroblasts and other nonimmune cells, and intrinsic pathway of apoptosis can be initiated by imbalance in the mitochondrial membrane potential Ym (Kulkarni et al., 1998). Mitochondrial control and guidance of cellular activities of T cells (Chao et al., 2017; O'Sullivan and Pearce, 2015) were recognized as a key factor in many diseases (Shinohara and Tsukimoto, 2018). In our study, cultivation of unstimulated splenocytes for 70 h decreased Ym and induced transition of naive splenocytes to early apoptosis (approximately 86%) and late apoptosis (approximately 11%). Activation with CoA stimulated Ym in T cells, which correlated with the decreased proportion of the early apoptotic cells (79%) indicating the metabolic reprogramming in activated cells. It was found that CoA bound to the cell membrane is internalized and accumulated primarily onto the mitochondria as early as 1 h posttreatment, and gradually increased mitochondrial membrane permeability. The increased mitochondrial membrane permeability would then lead to staining with Annexin V, the marker labeling the early stage, but no typical apoptosis was observed (Shinohara and Tsukimoto, 2018). We further showed that low concentrations of SIL (up to 10 μg/ml) positively modulated the mitochondrial dynamics in activated T cells which probably accounted for observed reduced proportions of the early apoptotic and late apoptotic/dead cells. Interestingly, higher concentrations (20 and 40 μg/ml) were not pro-apoptotic, in agreement with results of the study of Gharagozloo et al. (2010) on activated CD4+ T cells, supporting our hypothesis that antiproliferative effect of 40 μg/ml SIL (approximately 100 μM/ml) was not due to the cytotoxic effects on activated T cells. Detailed examination of the effect of SIL on proliferation revealed a significant G1 arrest in the cell cycle of CD3+ CD28+ activated T lymphocytes after 96 h of incubation with 100 μM SIL (approximately 50 μg/ml) without causing cell death (Gharagozloo et al., 2013). It is possible that the stimulated cells had an impaired respiratory capacity with a decreased mitochondrial membrane potential due to excess of produced reactive oxygen species. Addition of SIL to stimulated cells increased the mitochondrial membrane potential, suggesting that in CoA-stimulated cells the electron transfer, respiratory activity of the cells, and activity of the enzymes involved in oxidative phosphorylation have increased. Moreover, SIL is likely to prevent peroxidative damage of phospholipids, thereby preventing the initiation of apoptosis through cytochrome
In conclusion, our study demonstrated that mouse spleen T lymphocytes activated by lectin CoA show different susceptibilities to low (£10 μg/ml) and higher (20 and 40μg/ml) SIL concentrations after 70 h of incubation