Chrysophanol Mitigates T Cell Activation by Regulating the Expression of CD40 Ligand in Activated T Cells

International Journal of Molecular Sciences Article Chrysophanol Mitigates T Cell Activation by Regulating the Expression of CD40 Ligand in Activated T Cells Hyun-Su Lee and Gil-Saeng Jeong * College of Pharmacy, Keimyung University, Daegu 42601, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-(53)-580-6649 Received: 17 July 2020; Accepted: 24 August 2020; Published: 25 August 2020 Abstract: Since T lymphocytes act as mediators between innate and acquired immunity, playing a crucial role in chronic inflammation, regulation of T cell activation to suitable levels is important. Chrysophanol, a member of the anthraquinone family, is known to possess several bioactivities, including anti-microbial, anti-cancer, and hepatoprotective activities, however, little information is available on the inhibitory effects of chrysophanol on T cell activation. To elucidate whether chrysophanol regulates the activity of T cells, IL-2 expression in activated Jurkat T cells pretreated with chrysophanol was assessed. We showed that chrysophanol is not cytotoxic to Jurkat T cells under culture conditions using RPMI (Rosewell Park Memorial Institute) medium. Pretreatment with chrysophanol inhibited IL-2 production in T cells stimulated by CD3/28 antibodies or SEE-loaded Raji B cells. We also demonstrated that chrysophanol suppressed the expression of the CD40 ligand (CD40L) in activated T cells, and uncontrolled conjugation between B cells by pretreatment with chrysophanol reduced T cell activation. Besides, treatment with chrysophanol of Jurkat T cells blocked the NFκB signaling pathway, resulting in the abrogation of MAPK (mitogen-activated protein kinase) in activated T cells. These results provide novel insights into the suppressive effect of chrysophanol on T cell activation through the regulation of CD40L expression in T cell receptor-mediated stimulation conditions. Keywords: T cells; IL-2; CD40L; immunological synapse; MAPK 1. Introduction T cells play a critical role in inflammatory responses, wherein several immune cells are involved, and orchestrate the regulation of immune responses. Pathogenesis of various autoimmune disorders, including atopic dermatitis, immune bowel diseases, asthma, systemic lupus erythematosus, and psoriasis is implicated in the activated T cells [1]. Excessively activated T cells primed with self-antigens can lead to severe autoimmune responses. Several therapeutics for autoimmune disorders have been developed to control undesirable T cell activity in immune responses. Consequently, regulation of excessively activated T cells is pivotal for maintaining immunological homeostasis. CD40 ligand (CD40L) is a surface protein that is expressed on resting and activated T cells and shows induced patterns in activated T cells [2]. CD40L is known to be required for generating primary cytotoxic T cells in viral infections [3]. Deficiency or mutation of CD40L causes severe immunodeficiency and X-linked Hyper-IgM syndrome [4]. It binds to CD40 expressed on antigen presenting cells (APCs) in the early phase of immunological synapse (IS) and acts to transmit co-stimulatory signals to both T cells and APCs, which in turn help B cells proliferate and differentiate into plasma cells [5]. It is well known that sufficient signals between T cells and APCs promotes mature IS in the late phase of T cell activation. Although primed T cells from APCs are physically separated from T-APC conjugates Int. J. Mol. Sci. 2020, 21, 6122; doi:10.3390/ijms21176122 www.mdpi.com/journal/ijms Int.Int. J. Mol. Sci. Sci. 20192020,, 2021,, x 6122 22 of of 13 have explored whether CD40L plays a critical role in the separation of T cells in the late phase of T andcell activation, differentiated subsequently into effector leading T cells, to only the aregulation few investigators of T cell haveactivation. explored whether CD40L plays a criticalChrysophanol, role in the separation a phytochemical, of T cells in thealso late known phase ofas Tchrysophanic cell activation, acid, subsequently is a member leading of to the regulationanthraquinone of T cellfamily activation. that has been found in several plants, including Rheum palmatum [6], Cassia fistulaChrysophanol, [7], Aloe excels a [8], phytochemical, and Cluytia alsohirsute known [9]. It as possesses chrysophanic biological acid,is activities a member such of the as antitumor anthraquinone [10] familyand anti-diabetic that has been activities found in [11], several as well plants, as includingpreventiveRheum effects palmatum on memory[6], Cassia and fistula learning[7], Aloe functions excels [ 8in], andAlzheimer’sCluytia hirsute disease[9]. mouse It possesses model biological [12]. In activities particular, such anti-inflammatory as antitumor [10] and effects anti-diabetic of chrysophanol activities [ 11on], asdextran well as sulfate preventive sodium effects (DSS)-induced on memory and colitis learning and functionslipopolysaccharide in Alzheimer’s (LPS)-induced disease mouse inflammation model [12]. Inhas particular, been demonstrated anti-inflammatory to effectively effects ofsuppress chrysophanol overall on clinical dextran concentrations sulfate sodium of (DSS)-induced moieties, including colitis andthose lipopolysaccharide of interleukin-6 (IL- (LPS)-induced6), tumor necrosis inflammation factor- hasα (TNF- beenα demonstrated), and cyclooxygenase-2 to effectively (COX-2) suppress through overall clinicalthe regulation concentrations of NF ofκ moieties,B pathway including [13]. those Despite of interleukin-6 its protecti (IL-6),ve tumoractivity necrosis against factor- LPS-inducedα (TNF-α), andinflammation, cyclooxygenase-2 little is (COX-2) known through whether the chrysophanol regulation of NF hasκB a pathway suppressive [13]. Despiteeffect on its T protective cell activation. activity againstHere, we LPS-induced explored whether inflammation, chrysophanol little is known controls whether T cell chrysophanol activation mediated has a suppressive by T cell effectreceptors on T and cell activation.its underlying Here, mechanism we explored of whether action through chrysophanol the regulation controls T of cell CD40L activation expression mediated and by function. T cell receptors and its underlying mechanism of action through the regulation of CD40L expression and function. 2. Results 2. Results 2.1. Chrysophanol Is Not Cytotoxic to Jurkat T Cells under Culture Conditions Using RPMI Medium 2.1. Chrysophanol Is Not Cytotoxic to Jurkat T Cells under Culture Conditions Using RPMI Medium Chrysophanol (Figure 1), a member of the anthraquinone family, has been shown to possess Chrysophanol (Figure1), a member of the anthraquinone family, has been shown to possess anti-cancer activity, since it regulates proliferation and brings about apoptosis of cancerous cells [10]. anti-cancer activity, since it regulates proliferation and brings about apoptosis of cancerous cells [10]. In particular, chrysophanol has been reported to cause cytotoxicity and pro-apoptotic activities in In particular, chrysophanol has been reported to cause cytotoxicity and pro-apoptotic activities in Jurkat T cells cultured in DMEM (Dulbecco’s Modified Eagle Medium) medium [14]. By contrast, Jurkat T cells cultured in DMEM (Dulbecco’s Modified Eagle Medium) medium [14]. By contrast, several studies have reported that chrysophanol does not exhibit cytotoxic effects and protect cells several studies have reported that chrysophanol does not exhibit cytotoxic effects and protect cells from critical damages [15–17]. To clarify whether treatment with chrysophanol exhibits cytotoxicity from critical damages [15–17]. To clarify whether treatment with chrysophanol exhibits cytotoxicity on Jurkat T cells cultured using different conditions as previously reported, we performed an MTT on Jurkat T cells cultured using different conditions as previously reported, we performed an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay by comparing different media (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay by comparing different media (RPMI (Rosewell Park Memorial Institute) versus DMEM) and different densities of cells (2 × 104/mL (RPMI (Rosewell Park Memorial Institute) versus DMEM) and different densities of cells (2 104/mL to 1 × 105/mL). Figure 2A revealed that 40 μM chrysophanol did not exert cytotoxic effects ×on Jurkat to 1 105/mL). Figure2A revealed that 40 µM chrysophanol did not exert cytotoxic effects on Jurkat T cells× cultured in RPMI and DMEM at a density of 1 × 105/mL but displayed mild cytotoxicity to T cells cultured in RPMI and DMEM at a density of 1 105/mL but displayed mild cytotoxicity to Jurkat T cells cultured only in DMEM at a density of 5 ×× 104/mL or 2 × 104/mL. To obtain growth rate Jurkat T cells cultured only in DMEM at a density of 5 104/mL or 2 104/mL. To obtain growth of Jurkat T cells in the presence of 40 μM chrysophanol,× we counted the× number of Jurkat T cells rate of Jurkat T cells in the presence of 40 µM chrysophanol, we counted the number of Jurkat T cells cultured in these two media every 24 h. As shown in Figure 2B, Jurkat T cells cultured in DMEM cultured in these two media every 24 h. As shown in Figure2B, Jurkat T cells cultured in DMEM showed a significant decrease in growth rate compared to Jurkat T cells cultured in RPMI. To confirm showed a significant decrease in growth rate compared to Jurkat T cells cultured in RPMI. To confirm whether the population of apoptotic cells induced by treatment with chrysophanol is dependent on whether the population

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