Inhibitory effects of α-Mangostin on T cell cytokine secretion via ORAI1 calcium channel and K⁺ channels inhibition

Cell culture

Human embryonic kidney 293 T (HEK293T) and Jurkat T cells were purchased from the American Type Culture Collection (Manassas, VA, USA). HEK293T cells were cultured in a 10% CO₂ incubator at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM, Welgene, Gyeongsan, Korea). The culture medium contained 10% fetal bovine serum (FBS, Welgene) and 1% penicillin/streptomycin (P/S, Hyclone). Jurkat T cells were cultured in a 5% CO₂ incubator at 37 °C, in RPMI1640 medium (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS and 1% P/S.

Transient transfection

To measure the ORAI1 current, HEK293T cells were co-transfected with the human ORAI1 (hORAI1) and human STIM1 (hSTIM1) vector, which were purchased from Origene Technologies (Rockville, MD, USA). Transfection was performed using Turbofect (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s protocol, and green fluorescence protein (pEGFP-N1, Life Technologies) was injected at a 10:1 ratio for labeling of transfected cells.

Cell cytotoxicity

Cell viability was determined using the Cell Counting Kit 8 (CCK-8). Sample preparation and analysis were performed according to the manufacturer’s protocol. Jurkat T cells were prepared and 2 × 10⁴ cells/well were seeded in a 96-well microtiter plate, treated with α-mangostin 0.01 µM to 10 µM and incubated for 72 h. Following treatment, 10 µL of CCK-8 per 100 µL of culture medium was added into each well, incubated for 3 h at 37 °C, and the absorbance measured at 450 nm.

Electrophysiology

ORAI1 was measured using transiently transfected HEK293T cells, and the K_V1.3 current in Jurkat T cells was measured directly. A stable cell line, in which the corresponding ion channels were overexpressed, was used for K_Ca3.1. Recording and analysis of the whole-cell patch clamp for I_ORAI1 has been previously reported (Kim et al., 2018). The composition of the extracellular fluid for recording K_V1.3 current ( I_KV) and K_Ca3.1 current ( I_KCa) was 145 mM NaCl, 3.6 mM KCl, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 5 mM glucose, 1.3 mM CaCl₂, and 1 mM MgCl₂; pH 7.4 adjusted with NaOH. The composition of the internal solution was 5 mM NaCl, 140 mM KCl, 10 mM HEPES, 5 mM ethylene glycol-bis(β-aminoethyl ether)-N,N, N′, N′-tetraacetic acid, 2 mM Mg-ATP, 4.37 mM CaCl₂, and 0.5 mM MgCl₂; pH 7.2, adjusted with KOH. The intracellular calcium concentration for recording I_KCa was titrated to 1 µM and calculated using WEBMAXC (Stanford University, https://somapp.ucdmc.ucdavis.edu/pharmacology/bers/maxchelator/webmaxc/webmaxcS.htm). All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions and were prepared in dimethyl sulfoxide (DMSO). All stock solutions were stored at −20 °C.

Cytokine assay

Jurkat T cells were stimulated with anti-CD3 (Peprotech, Rocky Hill, NJ) and anti-CD28 (Peprotech) to induce the secretion of interleukin-2 (IL-2). Briefly, 50 µL/well of anti-CD3 at a concentration of 5 µg/mL was added to a 96-well plate, incubated at 37 °C for 3 h, and washed three times with Dulbecco’s phosphate-buffered saline (DPBS). Jurkat T cells were seeded at a density of 5 × 10⁵ cells/well. Thereafter 2 µg/mL anti-CD28 was added into each well and cultured in a 5% CO₂ incubator at 37 °C for 72 h. The culture solution was subsequently collected and diluted 1:3 with DMEM. The total amount of IL-2 secreted by Jurkat T cells was measured using the IL-2 ELISA kit (Peprotech) according to the manufacturer’s protocol.

Fura-2 Ca²⁺ imaging

[Ca²⁺]_i was measured using the fluorescent Ca²⁺ indicator, fura-2 acetoxymethyl ester (Fura-2 AM; Thermo Fisher Scientific). Jurkat T cells were incubated with normal Tyrode (NT) solution (145 mM NaCl, 10 mM HEPES, 5 mM glucose, 3.6 mM KCl, 2 mM CaCl₂, and 1 mM MgCl₂ ; pH 7.4, adjusted with NaOH), containing 2 µM Fura-2 AM for 30 min at 37 °C; and subsequently washed twice with NT. The cells were attached to a perfusion chamber with a 0.17 mm-thick glass bottom on an inverted microscope (Nikon eclipse Ti, Nikon, Osaka, Japan). Calcium signals were induced in the attached cells and the fluorescence was measured using an illuminator (pE-340 fura, CoolLED, Andover, UK) and recorded using a camera (sCMOS pco.edge 4.2, PCO, Kelheim, Germany). The cells were exposed to a flow excitation wavelength of 380 nm for 30 ms and an excitation wavelength of 340 nm for 100 ms; the emission was recorded at a wavelength of 510 nm. Images were recorded and were analyzed using NIS-Element AR Version 5.00.00 (Nikon).

Statistical analysis

Data analysis was carried out using GraphPad prism 6.0 (GraphPad) and Origin 8.0 (Microcal). Data were expressed as the mean  ± standard error of the mean (SEM). Bonferroni multiple comparison analysis was used following a one-way analysis of variance (ANOVA) multiple concentrations of components and IL-2 analysis. A p-value was <0.05 was considered statistically significant.

Inhibitory effect of α-mangostin on ORAI1

We measured I_ORAI1 using the whole-cell patch clamp technique following hORAI1 overexpression in HEK293T cells by transient transfection to determine whether α-mangostin inhibits I_ORAI1. Calcium stored in the ER to induce I_ORAI1 activity was depleted in IP₃ contained in the pipette solution. When the induced current was stable, it was treated with α-mangostin to confirm its inhibitory effect. α-Mangostin inhibited I_ORAI1 in a concentration-dependent manner (Figs. 1A–1B). Figure 1A shows the chart trace of I_ORAI1, and Fig. 1B shows the inhibition by α-mangostin as a current–voltage relationship curve. Figure 1C shows the I_ORAI1 half-maximal inhibitory concentrations (IC₅₀) of α-mangostin, with an IC₅₀ of 1.27 ± 1.144 µM. To confirm that ORAI1 inhibits intracellular calcium signaling, we measured the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in Jurkat T cells using the fluorescent dye Fura-2. To activate ORAI1, the ER was depleted using thapsigargin, an SERCA pump inhibitor. Upon changing from 0 Ca²⁺ to 2 mM Ca²⁺ following thapsigargin treatment, calcium influx by ORAI1 occurs. When calcium influx was maintained at a constant level, it was treated with 1 µM and 3 µM α-mangostin (Fig. 2A); [Ca²⁺]_i was inhibited by α-mangostin by 23.90 ± 12.501% and 77.14 ± 6.600%, respectively (Fig. 2B).

Inhibitory effect of α-mangostin on K_V1.3 and K_Ca3.1, the regulators of calcium signaling in immune cells

We measured the activity of potassium channels, which is necessary for maintaining the membrane driving force for sufficient calcium influx via ORAI1, and examined whether α-mangostin could inhibit it. It has been reported that K_V1.3 expression increases when the T-cell receptors (TCRs) of T-lymphocytes are stimulated (Decoursey et al., 1987). Therefore, Jurkat T cells were treated with 3 µg/mL anti-CD3 (Peprotech) for 24 h, and I_KV subsequently measured using the whole-cell patch clamp technique. When the cell membrane voltage was changed from -120 mV to +60 mV for 500 ms, I_KV increased from -60 mV (Fig. 3A). It was confirmed that I_KV decreased by 41.38 ± 6.191% at 3 µM (Fig. 3B) upon α-mangostin treatment when the magnitude of the current remained stable. Finally, it was confirmed that the current was completely reduced by treatment with PAP-1, an inhibitor of K_V1.3 (Fig. 3B). In addition, we examined the effect of α-mangostin on K_Ca3.1, which is activated when calcium signals are generated in T cells (Lam & Wulff, 2011). However, in Jurkat T cells, K_Ca3.1 expression was extremely low and the measured current was extremely small, which is not suitable for analyzing the inhibitory effect of α-mangostin. Therefore, whole-cell patch clamp was performed using the stable cell line overexpressing K_Ca3.1. As I_KCa is activated by an increase in intracellular calcium concentration, the intracellular calcium concentration was fixed at 1 µM. When I_KCa, activated by a fixed calcium concentration, was maintained at a constant level, it was inhibited by α-mangostin treatment in a concentration-dependent manner. As shown in Fig. 4A, α-mangostin inhibited I_KCa by 28.28 ± 5.412% and 51.16 ± 5.385% at concentrations of 1 µM and 3 µM, respectively. Normalized data are summarized in Fig. 4B.

Inhibitory effect of α-mangostin on IL-2 production in Jurkat T cells stimulated by anti-CD3/anti-CD28

Finally, we investigated whether α-mangostin inhibits cytokine secretion. First, the cytotoxicity of α-mangostin was evaluated. Following α-mangostin treatment of Jurkat T lymphocytes, cell viability was measured. Most cells died, when treated with α-mangostin at a concentration of 10 µM, however, >80% survived when treated with α-mangostin at a concentration of 3 µM (Fig. 5A). Thus, we subsequently analyzed whether the cytokine secretion of Jurkat T cells was inhibited at a maximum concentration of 3 µM or less. Consequently, α-mangostin inhibited IL-2 production in a concentration-dependent manner, and the highest inhibition rate (80.14 ± 3.987%) was observed at 3 µM (Fig. 5B).

Effects of α-mangostin on the calcium channel (ORAI1)

TCR stimulation by antigens activates T cells and triggers calcium signaling. This increased calcium is known to be involved in T cell proliferation and the production and secretion of cytokines (Feske, Wulff & Skolnik, 2015; Putney , 2012). TCR stimulation activates ORAI1, and the intracellular calcium increase by this process binds to Ca²⁺-modulated protein (calmodulin) to generate various downstream signals. Calcium-calmodulin activates the nuclear factor of activated T cells (NFAT), thereby regulating cytokine production and immune cell proliferation (Trebak & Kinet, 2019; Vaeth & Feske, 2018). Ion channels that generate calcium signals in immune cells include ORAI, transient receptor potential (TRP) channels, and voltage-dependent Ca²⁺ channels (TRP); however, the role of ion channels other than ORAI1 and STIM1 is controversial among researchers (Feske, Wulff & Skolnik, 2015; Kotturi, Hunt & Jefferies, 2006; Nohara et al., 2015; Stokes, MacKenzie & Sluyter, 2016). Functional impairment of ORAI1 or STIM1 deficiency causes severe combined immunodeficiency (SCID) in mouse models and human patients, and various functional problems have been reported (Prakriya & Lewis, 2015; Feske, Wulff & Skolnik, 2015; Nohara et al., 2015).

Therefore, I_ORAI1 was measured using HEK293T cells overexpressing hORAI1 and hSTIM1 to observe the pharmacological effects of α-mangostin. α-Mangostin suppressed most ORAI1 currents at a concentration of 10 µM, and demonstrated extremely high potency with an IC₅₀ of 1.27 ± 1.144 µM (Fig. 1).

Effects of α-mangostin on K⁺ channels (K_V1.3 and K_Ca3.1)

When Ca²⁺, which is a divalent cation, continuously flows into the cell, the cell membrane voltage is depolarized, and the driving force to introduce calcium is weakened. During this time, K_V1.3 and K_Ca3.1 maintain the driving force for calcium influx by maintaining a negative cell membrane potential in T cells (Cahalan & Chandy, 2009). They are known to be regulated by immunological synapses after TCR activation (Feske, Wulff & Skolnik, 2015). Inhibition of K_V1.3 and K_Ca3.1 has been reported to attenuate calcium, and consequently, are involved in the activity and proliferation of T cells as well as the production and proliferation of cytokines (Lam & Wulff, 2011; Koch Hansen et al., 2014; Feske, Skolnik & Prakriya, 2012). K_V1.3 and K_Ca3.1 are typically mentioned as ion channels that regulate calcium influx in T cells, and research is being conducted to develop new immunosuppressive agents using them (Lam & Wulff, 2011; Chandy & Norton, 2017). In addition to the inhibition of ORAI1, whether α-mangostin can inhibit K_V1.3 and K_Ca3.1, which controls calcium influx, were observed. As a result, 3 µM α-mangostin, that could inhibit 70% ORAI1 by approximately 70%, inhibited K_V1.3 by 41.38 ± 6.191%, and K_Ca3.1 by 51.16 ± 5.385%. Therefore, the inhibitory effect of α-mangostin on calcium influx appears to contribute to the inhibition of ORAI, as well as the inhibition of the potassium channels that regulate it.

Inhibitory effect of α-mangostin on calcium signaling

Intracellular calcium signaling depends on the inflow of calcium through ORAI1, and sufficient calcium inflow is achieved through hyperpolarization of the cell membrane by the activity of the K⁺ ion channel (Feske, Wulff & Skolnik, 2015; Cahalan & Chandy, 2009; Panyi, 2005). In Jurkat T cells, 3 µM α-mangostin inhibited thapsigargin-induced store-operated Ca²⁺ entry (SOCE) by 77.14 ± 6.600% (Fig. 2) and ORAI1 expression by 70.51 ± 6.185%. α-mangostin at this concentration showed a higher inhibition of calcium signaling, compared to inhibition of only ORAI1. This suggested that α-mangostin suppressed the calcium signaling more effectively through inhibition of both the K_V1.3 and K_Ca3.1 ion channels and ORAI1. Intracellular calcium signaling regulates the degranulation in mast cells. Calcium signaling by antigens in RBL-2H3 cells is inhibited by α-mangostin, possibly due to the inhibitory effect of α-mangostin on K_V1.3 and K_Ca3.1, and ORAI1 (Itoh et al., 2008).

Inhibitory effects of α-mangostin on cytokine production

Anti-CD3/anti-CD28 stimulation generates calcium signals and activates NFAT to promote T cell proliferation and production of IL-2. Thus, we investigated whether α-mangostin, with its inhibitory effects on ORAI1 and calcium signaling, could inhibit IL-2 production in Jurkat T cells. Prior to confirming the inhibition of IL-2 secretion, cytotoxicity of α-mangostin was first evaluated, and most of the cells died at a concentration of 10 µM. Therefore, we examined the inhibitory effects of α-mangostin on IL-2 production using concentrations up to 3 µM at which cells survived. At a concentration of 3 µM α-mangostin inhibited IL-2 secretion of Jurkat T cells stimulated with anti-CD3 and anti-CD28 by 80.14 ± 3.987% at a concentration of 3 µM. The results obtained earlier confirmed that 3 µM α-mangostin inhibited ORAI1 by approximately 70% and SOCE calcium signaling by approximately 77%, which is similar to the inhibition of IL-2 production observed with the same concentration of α-mangostin. Moreover, according to the results of a previous study on the inhibitory effects of α-mangostin on the secretion of cytokines in human peripheral blood mononuclear cells, α-mangostin was reported to inhibit IL-2 secretion by concanavalin A (ConA) stimulation. ConA is known to play a role in producing IL-2 by stimulating TCRs, leading to Ca²⁺ signaling and NFAT activation (Fujita et al., 2019; Kasemwattanaroj et al., 2013). This evidence indirectly supports the inhibitory effect on calcium signaling identified in this study.