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 of apoptotic cells induced by treatment with chrysophanol is dependent on culture media and cell number, AnnexinV/PI (Propidium Iodide) apoptosis assay was performed. culture media and cell number, AnnexinV/PI (Propidium Iodide) apoptosis assay was performed. Jurkat T cells cultured in DMEM exhibited an increased apoptotic population compared to Jurkat T Jurkat T cells cultured in DMEM exhibited an increased apoptotic population compared to Jurkat cells cultured in RPMI, however, treatment with chrysophanol has no pro-apoptotic at the density of T cells cultured in RPMI, however, treatment with chrysophanol has no pro-apoptotic at the density of 1 × 105/mL. These results suggest that chrysophanol does not cause cell death and apoptosis in Jurkat 1 105/mL. These results suggest that chrysophanol does not cause cell death and apoptosis in Jurkat T ×cells cultured in RPMI medium. T cells cultured in RPMI medium.

Figure 1. The chemical structure of chrysophanol. Figure 1. The chemical structure of chrysophanol.

Int.Int. J. J. Mol. Mol. Sci. Sci. 20192020, ,2021,, x 6122 33 of of 13

Figure 2. Chrysophanol is not cytotoxic to Jurkat T cells under culture condition using RPMI Figuremedium. 2. Chrysophanol (A) Cell viability is not cytotoxic of Jurkat to Jurkat T cells T cells treated under with culture the condition indicated using concentrations RPMI medium. of (chrysophanolA) Cell viability in of the Jurkat indicated T cells media treated at with the the indicated indicated density concentrations for 24 h wasof chrysophanol assessed by in MTT the indicated(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl media at the indicated density for tetrazolium 24 h was assessed bromide). by ( BMTT) The (3-(4,5-dimethylthiazol-2-yl)- growth rate of Jurkat T cells 2,5-diphenyltreated with /tetrazoliumwithout 40 µbromide).M chrysophanol (B) The growth in the indicatedrate of Jurkat media T cells at the treated indicated with/without density for40 μ theM chrysophanolindicated time in was the measured indicated bymedi cella counting.at the indicated (C) Apoptotic density for population the indicated of Jurkat time T was cells measured treated with by cell40 µ counting.M chrysophanol (C) Apoptotic in the indicated population media of Jurkat at the T indicated cells treated density with for 40 24 μ hM was chrysophanol acquired by in flow the indicatedcytometry. media The mean at the value indicated of three density experiments for 24 h wasSEM acquired (standard by error flow of cytometry. mean) is presented. The mean *valuep < 0.05 of ± threeversus experiments control (A,B )± orSEM between (standard two indicated error of mean) groups is (C presented.). CHR—chrysophanol, * p < 0.05 versus RPMI—Rosewell control (A,B Park) or betweenMemorial two Institute, indicated DMEM—Dulbecco’s groups (C). CHR—chrysophanol, Modified Eagle Medium. RPMI—Rosewell Park Memorial Institute, DMEM—Dulbecco’s Modified Eagle Medium. 2.2. Chrysophanol Inhibits IL-2 Production from Activated T Cells 2.2. ChrysophanolTo elucidate Inhibits whether IL-2 chrysophanol Production from suppresses Activated the T activity Cells of T cells in conditions of stimulation mediatedTo elucidate by T cell whether receptors chrysophanol (TCRs), the mRNA suppresses level oftheil2 activityand interleukin of T cells (IL-2), in conditions a marker of of stimulation early T cell mediatedactivation by produced T cell receptors from activated (TCRs), T the cells mRNA were determined.level of il2 and Pretreatment interleukin of (IL-2), Jurkat a Tmarker cells activated of early Tby cell immobilized activation CD3produced and CD28 from antibodies activated withT cells chrysophanol were determined. reduced Pretreatment the expression of ofJurkatil2 gene T cells in a activateddose-dependent by immobilized manner (FigureCD3 and3A). CD28 Moreover, antibodies the amountwith chrysophanol of IL-2 produced reduced from the activatedexpression T of cells il2 genewas alsoin a reduced dose-dependent in the presence manner of chrysophanol(Figure 3A). inMoreover, a time- or the dose-dependent amount of IL-2 manner produced (Figure from3B). activatedB cells, one T ofcells the was antigen-presenting also reduced in cells,the presence provide primingof chrysophanol signals that in a initiate time- or T celldose-dependent activation by mannerforming (Figure immunological 3B). B cells, synapses one of [18 the]. Toantigen-presenting confirm whether cells, chrysophanol provide priming also has signals a suppressive that initiate effect Ton cell T cellactivation activation by forming by interacting immunological with B cells, synapses Raji B[18]. cells To pulsed confirm with whether staphylococcal chrysophanol enterotoxin also has aE (SEE),suppressive a superantigen, effect on wereT cell used activation to activate by interacting Jurkat T cells. with Figure B cells,3C showsRaji B thatcells pre-treatment pulsed with staphylococcalwith chrysophanol enterotoxin hinders theE (SEE), mRNA a superantigen, level of il2 in Jurkatwere used T cells to conjugated activate Jurkat with T SEE-loaded cells. Figure Raji 3C B showscells in that a dose-dependent pre-treatment manner.with chrysophanol In Jurkat T cells hinders conjugated the mRNA with Rajilevel B cells,of il2 the in IL-2 Jurkat production T cells conjugatedwas gradually with downregulated SEE-loaded Raji depending B cells in ona dose the-dependent concentration manner. and incubation In Jurkat timeT cells of conjugated pretreated withchrysophanol Raji B cells, (Figure the IL-23D). production To address was whether gradua thelly downregulated inhibited T cell depending activity by on pre-treatment the concentration with and40 µ incubationM chrysophanol time of is pretreated reversed or chrysophanol recovered as (Fig theure level 3D). of To un-treated address cells,whether we the tested inhibited the removal T cell activityeffect of by chrysophanol pre-treatment after with pre-treatment 40 μM chrysophanol with chrysophanol is reversed byor comparingrecovered as mRNA the level level of un-treated of il2 from cells,activated we Ttested cells pre-treatedthe removal with effect chrysophanol of chrysoph for 30anol min after or 1 hpre-treatment only and activated with T chrysophanol cells post-washed by comparing mRNA level of il2 from activated T cells pre-treated with chrysophanol for 30 min or 1 h

Int.Int. J. J. Mol. Mol. Sci. Sci. 20192020, ,2021, ,x 6122 4 4of of 13 13 only and activated T cells post-washed with fresh media after pre-treated with chrysophanol for 30 minwith or fresh 1 h. mediaAs shown after in pre-treated Figure 3E, withpre-treatment chrysophanol with forchrysophanol 30 min or 1for h. 30 As min shown and 1 in h Figureof T cells3E, exertedpre-treatment significant with decrease chrysophanol of mRNA for 30 level min of and il2 but 1 h removal of T cells of exerted chrysphanol signiﬁcant after decrease pre-treatment of mRNA did il2 notlevel show of anybut removalattenuation of chrysphanolin mRNA level after of pre-treatment il2. Interestingly, did not decrease show any of mRNA attenuation level in of mRNA il2 is il2 il2 dependentlevel of .on Interestingly, the time of decrease pre-treatment of mRNA with level chrysophanol, of is dependent meaning on thethat time pre-treatment of pre-treatment with chrysophanolwith chrysophanol, for 1 h meaning exhibited that more pre-treatment inhibitory witheffect chrysophanol on T cell activation for 1 h than exhibited pre-treatment more inhibitory for 30 min.eﬀect These on T data cell imply activation that T than cell activity pre-treatment is irreversibly for 30 min.restrained These by data pretreatment imply that with T cellchrysophanol activity is inirreversibly Jurkat T cells restrained stimulated by pretreatment by antibodies with against chrysophanol CD3/CD28 in and Jurkat SEE-pulsed T cells stimulated Raji B cells. by antibodies against CD3/CD28 and SEE-pulsed Raji B cells.

Figure 3. Chrysophanol inhibits IL-2 production from activated T cells. (A,C) Jurkat T cells were Figurepre-treated 3. Chrysophanol with the indicated inhibits concentrations IL-2 produc oftion chrysophanol from activated for 1 T h cells. and stimulated (A,C) Jurkat with T anti-CD3cells were/CD28 pre- treatedantibodies with (A the) or indicated SEE-loaded concentrations Raji B cells ( Cof) chryso for 6 h.phanol Induced for mRNA 1 h and levels stimulated of il2 gene with were anti-CD3/CD28 determined antibodiesby conservative (A) or PCR SEE-loaded (top) and quantitative Raji B cells PCR (C) (bottom). for 6 h. (BInduced,D) Jurkat mRNA T cells levels were pre-treated of il2 gene with were the determinedindicated concentrations by conservative (left) PCR or 40 (topµM) and (right) quantitative of chrysophanol PCR (bottom). for 1 h and (B activated,D) Jurkat by T anti-CD3cells were/CD28 pre- treatedantibodies with ( Bthe) or indicated SEE-loaded concentrations Raji B cells (left) (D) or for 40 24 μM h (left)(right) or of 0–24 chrysophanol h (right). for Produced 1 h and IL-2activated from byactivated anti-CD3/CD28 Jurkat T cellsantibodies was measured (B) or SEE-loaded by ELISA. (RajiE) Jurkat B cells T ( cellsD) for were 24 pre-treatmenth (left) or 0–24 with h (right). 40 µM Producedchrysophanol IL-2 for from 30 minactivated or 1 h andJurkat washed T cells with was fresh measured media. Cellsby ELISA. were stimulated(E) Jurkat withT cells anti-CD3 were pre- and treatmentCD28 antibodies with 40 for μM 6 h chrysophanol then mRNA level for 30 of il2minwas or assessed1 h and bywa quantitativeshed with fresh PCR. media. The mean Cells value were of stimulatedthree experiments with anti-CD3SEM is and presented. CD28 anti * p

Int. J. Mol. Sci. 2020, 21, 6122 5 of 13 Int. J. Mol. Sci. 2019, 20, x 5 of 13

cytometry analyses were performed. Western blotting results exhibited that pretreatment with flow cytometry analyses were performed. Western blotting results exhibited that pretreatment with chrysophanol downregulated CD40L expression in whole lysates of T cells in a dose-dependent chrysophanol downregulated CD40L expression in whole lysates of T cells in a dose-dependent manner manner (Figure 4B). Besides, the suppressive effect of chrysophanol on induction of CD40L (Figure4B). Besides, the suppressive e ffect of chrysophanol on induction of CD40L expression on expression on the surface of T cells was assessed by flow cytometry depending on stimulation time the surface of T cells was assessed by flow cytometry depending on stimulation time (Figure4C). (Figure 4C). The highest expression of CD40L was observed in T cells stimulated for 24 h. These data The highest expression of CD40L was observed in T cells stimulated for 24 h. These data suggest that suggest that the expression of CD40L on activated T cells is gradually increased by stimulation with the expression of CD40L on activated T cells is gradually increased by stimulation with CD3 and CD28 CD3 and CD28 antibodies, but pretreatment with chrysophanol inhibits CD40L expression in the antibodies, but pretreatment with chrysophanol inhibits CD40L expression in the whole lysate and on whole lysate and on surfaces of T cells. surfaces of T cells.

Figure 4. Chrysophanol suppresses the expression of CD40L in activated T cells. (A) Jurkat T cells were Figure 4. Chrysophanol suppresses the expression of CD40L in activated T cells. (A) Jurkat T cells pre-treated with 40 µM of chrysophanol for 1 h and stimulated by anti-CD3/CD28 antibodies for 3 to 48 h. were pre-treated with 40 μM of chrysophanol for 1 h and stimulated by anti-CD3/CD28 antibodies mRNA levels of cd40l were determined by conservative (top) and quantitative (bottom) PCR. (B) Jurkat for 3 to 48 h. mRNA levels of cd40l were determined by conservative (top) and quantitative (bottom) T cells were pre-treated with the indicated concentrations of chrysophanol for 1 h and stimulated by PCR. (B) Jurkat T cells were pre-treated with the indicated concentrations of chrysophanol for 1 h and anti-CD3/CD28 antibodies for 24 h. The expressed CD40Ls were detected by Western blot analysis, stimulated by anti-CD3/CD28 antibodies for 24 h. The expressed CD40Ls were detected by Western normalized to β-actin, and presented in a bar graph. (C) Jurkat T cells were pre-treated with 40 µM of blot analysis, normalized to β-actin, and presented in a bar graph. (C) Jurkat T cells were pre-treated chrysophanol for 1 h and stimulated by anti-CD3/CD28 antibodies for 12 to 48 h. The expressions of with 40 μM of chrysophanol for 1 h and stimulated by anti-CD3/CD28 antibodies for 12 to 48 h. The CD40L on cell surface were acquired by flow cytometry. Mean fluorescence intensities were presented in expressions of CD40L on cell surface were acquired by flow cytometry. Mean fluorescence intensities line graph. The mean value of three experiments SEM is presented. * p < 0.05 between cells stimulated were presented in line graph. The mean value± of three experiments ± SEM is presented. * p < 0.05 by anti-CD3/CD28 and cells indicated. CHR—chrysophanol, MFI—Mean fluorescence intensity. between cells stimulated by anti-CD3/CD28 and cells indicated. CHR—chrysophanol, MFI—Mean 2.4. Abnormalfluorescence Expression intensity. of CD40L by Chrysophanol Reduces T Cell Activation through Uncontrolled Conjugation between T and B Cells 2.4. Abnormal Expression of CD40L by Chrysophanol Reduces T Cell Activation through Uncontrolled Since expressed CD40L on naïve T cells provides co-stimulatory signaling for the initiation Conjugation between T and B Cells of T cell activation, we investigated whether chrysophanol affects the formation of conjugates of T cellsSince with Bexpressed cells in the CD40L early on phase naïve (30 T min)cells provides and late phaseco-stimulatory (24 h) of Tsignaling cell activation. for the Theinitiation highest of T conjugationcell activation, between we investigated T and B cells whether was observed chrysophanol within affects 30 min the but formation no significant of conjugates alteration of due T cells to pretreatmentwith B cells with in chrysophanolthe early phase was (30 observed min) and in cells late incubated phase (24 for h) 30 of min T (Figurecell activation.5A,B). Conjugation The highest wasconjugation also detected between in cells T and incubated B cells for was 24 observed h but was within downregulated 30 min but compared no significant to cells alteration incubated due for to 30pretreatment min. However, with enhanced chrysophanol conjugation was wasobserved observed in incells cells incubated incubated for for 2430 hmin in the (Figure presence 5A,B). of chrysophanolConjugation than was inalso control detected cells. in To cells examine incubated whether for 24 diff herent but was induction downregulated of CD40L oncompared the surface to cells of Tincubated cells by pretreatment for 30 min. However, with chrysophanol enhanced leadsconjugation to the incrementwas observed of conjugation in cells incubated in cells for pretreated 24 h in the presence of chrysophanol than in control cells. To examine whether different induction of CD40L on the surface of T cells by pretreatment with chrysophanol leads to the increment of conjugation in cells

Int. J. Mol. Sci. 2019, 20, x 6 of 13 Int. J. Mol. Sci. 2020, 21, 6122 6 of 13 pretreated with chrysophanol, CD40L antibodies were added at the initiation of incubation to withneutralize chrysophanol, CD40L. Interestingly, CD40L antibodies conjugation were added was at significantly the initiation enhanced of incubation in cells to neutralize incubated CD40L. with CD40LInterestingly, antibodies conjugation and reached was significantly comparable enhanced levels as in that cells in incubated cells pretreated with CD40L with antibodieschrysophanol and (Figurereached 5A,B). comparable To understand levels as that the ineffect cells of pretreated CD40L ex withpression chrysophanol on T cell activation, (Figure5A,B). the To production understand of IL-2the e ffwasect ofassessed CD40L expressionby conjugating on T cellT cells activation, with B the cells production and incubating of IL-2 was with assessed CD40L by neutralizing conjugating Tantibodies. cells with BThe cells amount and incubating of IL-2 produced with CD40L was neutralizing suppressed antibodies. in T-B conjugates The amount treated of IL-2 with produced CD40L wasneutralizing suppressed antibodies, in T-B conjugates and this treated inhibitory with CD40L effect neutralizing was similar antibodies, to that andof thispretreatment inhibitory ewithffect waschrysophanol similar to (Figure that of pretreatment 5C). Microscopic with chrysophanolimages were obtained (Figure5 C).using Microscopic an IncuCyte images imaging were system obtained to confirmusing an whether IncuCyte restricted imaging systemexpression to confirm of CD40L whether by chrysophanol restricted expression affects the ofCD40L conjugation by chrysophanol between T andaffects B cells the conjugation (Figure 5D,E). between These Tdata and imply B cells that (Figure the 5inducedD,E). These CD40L data on imply T cell that surface the induced plays a CD40Lpivotal roleon T in cell the surface regulation plays of a immunological pivotal role in thesynapses regulation in terminal of immunological phase. However, synapses pretreatment in terminal of T phase. cells withHowever, chrysophanol pretreatment fails to of regulate T cells with conjugation chrysophanol of T and fails B cells to regulate by downregulating conjugation ofCD40L T and expression B cells by indownregulating T cells, resulting CD40L in the expression reduction inof TT cells,cell activation. resulting in the reduction of T cell activation.

Figure 5. Abnormal expression of CD40L by chrysophanol leads to reduction of T cell activation Figure 5. Abnormal expression of CD40L by chrysophanol leads to reduction of T cell activation through uncontrolled conjugation between B cells. (A–E) CMFDA (5-chloromethylﬂuorescein diacetate) through uncontrolled conjugation between B cells. (A–E) CMFDA (5-chloromethylfluorescein stained Jurkat T cells were pre-treated with 40 µM of chrysophanol for 1 h and mixed with CMRA diacetate) stained Jurkat T cells were pre-treated with 40 μM of chrysophanol for 1 h and mixed with (5-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine) stained Raji B cells pulsed with SEE CMRA (5-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine) stained Raji B cells pulsed with (staphylococcal enterotoxin E) for 30 min or 24 h. In some cases, anti-CD40L neutralizing antibodies SEE (staphylococcal enterotoxin E) for 30 min or 24 h. In some cases, anti-CD40L neutralizing (20 µg/mL) were added to Jurkat T cells. Conjugation was determined by ﬂow cytometry (A) and antibodies (20 μg/mL) were added to Jurkat T cells. Conjugation was determined by flow cytometry double-positive population was presented in bar graph (B). Produced IL-2 from conjugates was assessed (A) and double-positive population was presented in bar graph (B). Produced IL-2 from conjugates by ELISA (C). Cells were seeded in plates and microscopic images of conjugates between T cells and B was assessed by ELISA (C). Cells were seeded in plates and microscopic images of conjugates between cells were obtained by IncuCyte® imaging system. White arrows indicate conjugates between T and B T cells and B cells were obtained by IncuCyte® imaging system. White arrows indicate conjugates cells (D). The ratio of conjugated T cells was calculated in percentages of total T cells (E). The mean between T and B cells (D). The ratio of conjugated T cells was calculated in percentages of total T cells value of three experiments SEM (standard error of mean) is presented. * p < 0.05 between the two (E). The mean value of three± experiments ± SEM (standard error of mean) is presented. * p < 0.05 groups indicated. CHR—chrysophanol. between the two groups indicated. CHR—chrysophanol.

2.5. Treatment with Chrysophanol Blocks NFκB Signaling Pathway in Activated T Cells Among the several signaling pathways associated with IL-2 production in activated T cells, the NFκB pathway has been considered as one of the central modulators. To examine whether the

Int. J. Mol. Sci. 2020, 21, 6122 7 of 13

2.5. Treatment with Chrysophanol Blocks NFκB Signaling Pathway in Activated T Cells Int. J. AmongMol. Sci. 2019 the, 20 several, x signaling pathways associated with IL-2 production in activated T7 cells,of 13 the NFκB pathway has been considered as one of the central modulators. To examine whether the inhibitory effect of chrysophanol on T cell activation is involved in the NFκB pathway, p65 inhibitory eﬀect of chrysophanol on T cell activation is involved in the NFκB pathway, p65 translocation translocation was determined in activated T cells pretreated with chrysophanol. Western blot results was determined in activated T cells pretreated with chrysophanol. Western blot results revealed that revealed that p65 in the cytosol was translocated by stimulation with CD3/CD28 antibodies, but p65 in the cytosol was translocated by stimulation with CD3/CD28 antibodies, but pretreatment with pretreatment with chrysophanol hindered this manifestation (Figure 6A). Degradation of IκBα by T chrysophanol hindered this manifestation (Figure6A). Degradation of I κBα by T cell activation was cell activation was also suppressed due to pretreatment with chrysophanol. Besides, pretreatment also suppressed due to pretreatment with chrysophanol. Besides, pretreatment with chrysophanol with chrysophanol downregulated the phosphorylation level of IκBα in activated T cells. These data downregulated the phosphorylation level of IκBα in activated T cells. These data suggest that suggest that chrysophanol blocks the NFκB pathway in activated T cells. chrysophanol blocks the NFκB pathway in activated T cells.

Figure 6. Treatment with chrysophanol blocks NFκB signaling pathway in activated T cells. (A) Jurkat Figure 6. Treatment with chrysophanol blocks NFκB signaling pathway in activated T cells. (A) Jurkat T cells were pre-treated with 40 µM of chrysophanol for 1 h and stimulated by anti-CD3/CD28 T cells were pre-treated with 40 μM of chrysophanol for 1 h and stimulated by anti-CD3/CD28 antibodies for 1 h. Translocated p65, IκBα and phosphorylated IκBα were analyzed by Western antibodies for 1 h. Translocated p65, IκBα and phosphorylated IκBα were analyzed by Western blot blot from nuclear and cytosolic extracts. (B) Detected bands were normalized to PARP (poly from nuclear and cytosolic extracts. (B) Detected bands were normalized to PARP (poly (ADP-ribose) (ADP-ribose) polymerase) for p65 in nuclear extract and β–actin for p65 in cytosolic extract, IκBα and polymerase) for p65 in nuclear extract and β–actin for p65 in cytosolic extract, IκBα and phosphorylated IκBα. Normalization was shown in bar graph. The mean value of three experiments phosphorylated IκBα. Normalization was shown in bar graph. The mean value of three experiments SEM is presented. * p < 0.05 between cells stimulated by anti-CD3/CD28 and cells indicated. ±± SEM is presented. * p < 0.05 between cells stimulated by anti-CD3/CD28 and cells indicated. CHR— CHR—chrysophanol, Con—control. chrysophanol, Con—control. 2.6. Pre-Treatment with Chrysophanol Abrogates MAPK Signaling Pathway in Activated T Cells 2.6. Pre-Treatment with Chrysophanol Abrogates MAPK Signaling Pathway in Activated T Cells To investigate whether chrysophanol suppresses the MAPK (mitogen activated protein kinase) signalingTo investigate pathway inwhether activated chrysophanol T cells, phosphorylation suppresses the levels MAPK of ERK (mitogen (extracellular activated signal-regulated protein kinase) kinases),signaling p38, pathway JNK (c-Junin activatedN-terminal T cells, kinases), phosphorylation and c-Jun werelevels detected of ERK (extracellular in time- and dose-signal-regulated dependent manner.kinases), Phosphorylation p38, JNK (c-Jun levelsN-terminal of ERK, kinases), p38, JNK, and and c-Jun c-Jun were were detected upregulated in time- by CD3and /dose-CD28 dependent antibodies withinmanner. 30 min,Phosphorylation but pretreatment levels with of chrysophanolERK, p38, JNK, partially and abrogatedc-Jun were these upregulated levels in activated by CD3/CD28 T cells (Figureantibodies7A). within A dose-dependent 30 min, but experimentpretreatment also with demonstrated chrysophanol that partially pretreatment abrogated with chrysophanolthese levels in exhibitsactivated impeded T cells phosphorylation(Figure 7A). A dose-dependent of ERK, p38, JNK andexperiment c-Jun in activatedalso demonstrated T cells (Figure that7B). pretreatment These data implywith chrysophanol that the MAPK exhibits pathway impeded is involved phosphorylation in suppressive of ERK, eﬀects p38, of chrysophanolJNK and c-Jun on in Tactivated cell activation T cells by(Figure CD3/ CD287B). These antibodies. data imply that the MAPK pathway is involved in suppressive effects of chrysophanol on T cell activation by CD3/CD28 antibodies.

Int. J. J. Mol. Mol. Sci. Sci. 20192020,, 2021,, x 6122 88 of of 13

Figure 7. Pre-treatment with chrysophanol abrogates MAPK (mitogen activated protein kinase) Figuresignaling 7. pathwayPre-treatment in activated with Tchrysophanol cells. (A,B) Jurkat abrogates T cells MAPK were pre-treated (mitogen with activated 40 µM ofprotein chrysophanol kinase) signalingfor 1 h and pathway stimulated in activated by anti-CD3 T cells/CD28. (A antibodies,B) Jurkat forT cells 15 and were 30 minpre-treated (A). Jurkat with T 40 cells μM were of chrysophanolpre-treated with for the 1 indicatedh and stimulated concentrations by anti-CD3/CD28 of chrysophanol antibodies for 1 h and for stimulated15 and 30 bymin anti-CD3 (A). Jurkat/CD28 T cellsantibodies were forpre-treated 30 min (B with). The the phosphorylated indicated concentrations levels of the of indicated chrysophanol proteins for were 1 h detected and stimulated by Western by anti-CD3/CD28blot analysis and antibodies normalized for to30 themin indicated (B). The phosphorylated total proteins. The levels mean of the value indicated of three proteins experiments were detectedSEM is by presented. Western blot * p analysis< 0.05 betweenand normalized cells stimulated to the indicated by anti-CD3 total proteins./CD28 and The cellsmean indicated. value of ± threeCHR—chrysophanol, experiments ± SEM ERK—extracellular is presented. * p < signal-regulated 0.05 between cells kinases. stimulated by anti-CD3/CD28 and cells indicated. CHR—chrysophanol, ERK—extracellular signal-regulated kinases. 3. Discussion 3. Discussion In the present study, we explored whether chrysophanol, a member of the anthraquinone family, has anIn inhibitorythe present e study,ffect on we TCR-mediated explored whether activation chryso ofphanol, T cells a bymember using of Jurkat the anthraquinone T cells. Pretreatment family, withhas an chrysophanol inhibitory effect effectively on TCR-mediated regulated IL-2 activation production of T incells activated by using T cellsJurkat without T cells. displaying Pretreatment any withcytotoxic chrysophanol effects under effectively culture regu conditionlated IL-2 using production RPMI medium. in activated In the T late cells phase without of T displaying cell activation, any cytotoxicexpression effects of CD40L under was culture downregulated condition using by pretreatment RPMI medium. of activatedIn the late T phase cells withof T cell chrysophanol. activation, expressionThis treatment of CD40L inhibited was the downregulated separation of Tby cells pretre fromatment IS in of the activated late phase T cells of conjugation with chrysophanol. of T cells withThis treatment B cells, thereby inhibited suppressing the separation T cell activation.of T cells fr Furthermore,om IS in the late results phase of Western of conjugation blotting of analysis T cells withindicated B cells, that thereby the inhibitory suppressing effect T cell of chrysophanol activation. Furthermore, on T cell activation results of was Western highly blotting mediated analysis by a indicateddecreasein that NF κtheB andinhibitory MAPK effect pathways. of chrysophanol on T cell activation was highly mediated by a decreaseIt is wellin NF knownκB and that MAPK cell viability pathways. assays including MTT and CCK-8 (cell counting kit-8) assays are highlyIt is dependent well known on thethat number cell viability of seeded assays cells including and culture MTT conditions and CCK-8 including (cell counting the types kit-8) of assays media areused highly and presence dependent or absence on the ofnumber other agents of seeded such ascell fetals and bovine culture serum conditions (FBS). RPMI including is known the totypes have of a medialower concentrationused and presence of calcium or absence than DMEM. of other Thisagents culture such conditionas fetal bovine is more serum suitable (FBS). for RPMI suspension is known cell tocultures have a such lower as thatconcentration of Jurkat T of cells calcium or Raji than B cells. DMEM. Nevertheless, This culture a previous condition study is more has reported suitablethat for suspensionchrysophanol cell exhibits cultures cytotoxic such as ethatffects of and Jurkat promotes T cells anor apoptoticRaji B cells. pathway Nevertheless, in Jurkat a Tprevious cells cultured study hasin DMEM reported medium that chrysophanol [14]. To check exhibits this discrepancy, cytotoxic effects we compared and promotes our culture an apoptotic conditions pathway to those in Jurkatdescribed T cells in previouscultured reports.in DMEM We medium found that [14]. Yin To Jcheck and colleagues this discrepancy, cultured we Jurkat compared T cells our in DMEMculture conditionsmedium, performed to those described cytotoxicity in previous assays with reports. different We densitiesfound that of Yin cells, J and and colleagues obtained Jurkat cultured cells Jurkat from Tdi ffcellserent in companies. DMEM medium, We cultured performed Jurkat cytotoxicity T cells in RPMI assays medium with accordingdifferent densities to the manufacturer’s of cells, and obtainedinstructions Jurkat and cells performed from different experiments companies. at a density We cultured of 1 Jurkat104/200 T µcellsL/well. in RPMI Under medium these conditions, according × weto the assessed manufacturer’s whether treatment instructions with and 40 µ peMrformed chrysophanol experiments for 24 h at has a cytotoxicdensity of e ff1ects × 10 in4/200 Jurkat μL/well. T cells Underand performed these conditions, several experiments we assessed to whether demonstrate treatment the regulatory with 40 μ effMect chrysophanol of chrysophanol for 24 on h T has cell cytotoxicactivity. The effects data in thus Jurkat obtained T cells suggest and performed that chrysophanol several experiments not only has to anticancer demonstrate activities the regulatory in various effectcells, butof chrysophanol also shows anti-inflammatory on T cell activity. e ffTheects data on several thus obtained cell lines, suggest including that Jurkat chrysophanol T cells used not in only the haspresent anticancer study. activities in various cells, but also shows anti-inflammatory effects on several cell lines, including Jurkat T cells used in the present study.

Int. J. Mol. Sci. 2020, 21, 6122 9 of 13

IS shown to be a distinct structure wherein several molecules play a critical role in T cell and APC activation [19]. A central supramolecular activation cluster (cSMAC) has been identiﬁed to be the most important part of IS in which pivotal molecules, including TCR, Lck, ZAP70, PKCθ, and CD28, accumulate for recognition of peptide-MHCII complex. CD40L is shown to be localized in the cSMAC of resting T cells and ligates with CD40 expressed on APCs [19]. A recent study demonstrated that CD40L-CD40 conjugation in cSMAC is dependent on the presence of TCR signaling, binding of intracellular adhesion molecule 1 (ICAM-1) to integrin molecules expressed on T cells, and rearrangement of the cytoskeleton in T cells [19]. Although evidence explaining the mechanism of IS termination has been demonstrated, depletion of TCR recycling process induces separation of T cells from conjugation [20] and blockade of CD40L-controlled termination of immune synapse formation (Figure5). This result assumes that conjugation between T cells and APCs is tightly regulated so as not to be activated by the induction of CD40L in the late phase of T cell activation. Furthermore, pretreatment with chrysophanol suppressed the expression of CD40L in activated T cells and abolished termination of contact between T cells and APCs. Based on these results, the present study implies that chrysophanol has a potential to be developed as an immunosuppressive agent by targeting CD40L. To activate resting T cells in vitro, a direct stimulation method mediated by TCR is widely used. A mixture of superantigen-loaded B cells and T cells is commonly used to stimulate T cells in an in vitro experiment [21]. It has been developed to mimic the actual situation where TCR engages peptide-MHC complex from B cells in the IS. In this procedure, in vitro immune synapse formation can be assessed using Jurkat T cells and SEE-loaded Raji B cells [22]. The other method to provide T cell-activating signals is to use monoclonal antibodies against CD3 and CD28 constituting TCR. Immobilized CD3 antibodies are potent stimulators that target CD3 without employing the peptide-MHC complex [23]. To supply co-stimulatory signals with CD3 stimulation, soluble CD28 antibodies are orchestrated. The current study revealed that Jurkat T cells were activated by SEE-loaded Raji B cells and antibodies against CD3 and CD28. A tremendous increment of IL-2 levels and phosphorylated MAPK signaling molecules from activated Jurkat T cells demonstrated that the in vitro stimulatory method mediated by TCR triggers phosphorylation of ERK, p38, JNK, and c-Jun, in turn promoting the production of IL-2. To investigate whether chrysophanol has a suppressive eﬀect on T cell activation in vivo, further studies should investigate if oral administration of chrysophanol attenuates manifestations of T cell-mediated diseases such as atopic dermatitis, immune bowel disease, or asthma using an animal model.

4. Materials and Methods

4.1. Cells Jurkat T cells and Raji B cells were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea). Cells were grown in RPMI medium (Welgene, Gyeongsan, Republic of Korea) supplemented with 10% fetal bovine serum (FBS), streptomycin (100 µg/mL), penicillin G (100 units/mL) and l-glutamine (2 mM). Cells were grown at 37 ◦C in a humidiﬁed incubator containing 5% CO2 and 95% air.

4.2. Reagents and Antibodies MTT (1-(4,5-Dimethylthiazol-2-yl)-3,5-diphenylformazan) powder, CMFDA (5-chloromethylfluorescein diacetate) and CMRA (5-(((4-chloromethyl)benzoyl)amino) tetramethylrhodamine) cell staining dyes, was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Anti-CD3 antibodies and anti-CD28 antibodies were purchased from Bioxcell (West Lebanon, NH, USA). Human IL-2 DuoSet® ELISA kit was obtained from R&D Systems (Minneapolis, MN, USA). Staphylococcal enterotoxin E (SEE) was purchased from Toxin Technology (Sarasota, FL, USA). NE-PER Nuclear and Cytoplasmic Extraction Reagents Kit and ECL Western blotting detection reagents were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Anti-CD40L antibodies conjugated with APC was purchased from eBiosciences (San Diego, CA, USA). Int. J. Mol. Sci. 2020, 21, 6122 10 of 13

Anti-CD40L neutralizing antibodies were obtained from InvivoGen (San Diego, CA, USA). Anti-CD40L for western blot and anti-β-actin antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-p65, anti-PARP, anti-IκBα, anti-phosphorylated IκBα (S32), anti-phosphorylated ERK (T202/Y204), anti-ERK, anti-phosphorylated p38 (T180/Y182), anti-p38, anti-phosphorylated JNK (T183/Y185), anti-JNK, anti-phosphorylated c-Jun (S73) and anti-c-Jun antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).

4.3. Isolation of Chrysophanol from Rumex crispus L Chysophanol used in the current study was isolated from dried leaves of R. crispus L. as previously reported in our group [24]. Briefly, dried leaves of R. crispus L. (1 kg) were extracted 3 times with 70% ethanol for 24 h under room temperature. The EtOH extract (230 g) was suspended in H2O (1 L) and then partitioned with n-hexane (1 L) and EtOAc (1 L). Among the n-hexene (4.5 g) and EtOAc fraction (27.1 g), chrysophanol was purified by repeated open column chromatography on silica gel (6.5 60 cm; 70–230 mesh). The isolated chrysophanol was analyzed by 1H and 13C-NMR × (JEOL JNM-ECA 500) NMR spectroscopic, and spectral data were identified as chrysophanol by comparison with the published literature [25].

4.4. MTT Assay Two diﬀerent media (RPMI or DMEM) cultured-Jurkat T cells (1 105–2 104/mL) were seeded × × and incubated with the indicated concentration of chrysophanol (0–40 µM) for 24 h. The supernatants were discarded and cells were added with 500 µg/mL of MTT for 1.5 h. Supernatants were removed and 150 µL of DMSO were added for dissolving formazan crystals. Absorbance was obtained at 540 nm and cell viability was calculated with absorbance of control (% of control).

4.5. Assessment of Growth Rate by Cell Counting Two diﬀerent media (RPMI or DMEM) cultured-Jurkat T cells (1 10–2 104/mL) were seeded × × and incubated with 40 µM chrysophanol for the indicated time. The number of cells was counted every 24 h and was presented in line graph.

4.6. AnnexinV/PI Apoptosis Assay by Flow Cytometry Two diﬀerent media (RPMI or DMEM) cultured-Jurkat T cells (1 105–2 104/mL) were seeded × × and incubated with the indicated concentration of chrysophanol (0–40 µM) for 24 h. After incubation, cells were stained with AnnexinV and PI and apoptotic cells were acquired by ﬂow cytometry. The population of apoptotic cells were presented in dot plots.

4.7. T Cell Stimulation Jurkat T cells (5 105/mL) were pre-treated with chrysophanol at the indicated concentration × (0–40 µM) or 40 µM for 1 h at 37 ◦C. Then cells were stimulated with plates coated with anti-CD3 (20 µg/mL) and soluble anti-CD28 (7 µg/mL) for the indicated time (6–24 h). For superantigen stimulation, Raji B cells were prepared by pulsing 1 µg/mL SEE for 30 min at 37 ◦C. Then Jurkat T cells were co-cultured with SEE-pulsed Raji B cells for the indicated time (6–24 h).

4.8. Determination of mRNA Level by Conventional and Quantitative PCR Total RNA was isolated using TRIZOL reagent (Thermo Fisher Scientiﬁc, Waltham, MA, USA) and reverse transcription of the RNA to cDNA was performed using RT PreMix (Enzynomics, Daejeon, Korea). Primers used for each gene were as follows (forward and reverse primers, respectively): human il2, 50-CAC GTC TTG CAC TTG TCA C-30 and 50-CCT TCT TGG GCA TGT AAA ACT-30; human cd40l, 50-GCC AGT TTG AAG GCT TTG TG-30 and 50-ACT TAT GAC ATG TGC CGC AA-30, human gapdh, 50-CGG AGT CAA CGG ATT TGG TCG TAT-30 and 50-AGC CTT CTC CAT GGT GGT GAA Int. J. Mol. Sci. 2020, 21, 6122 11 of 13

GAC-30. The condition of conventional PCR was as follows: 28 cycles of denaturation at 94 ◦C for 30 s, annealing at 60 ◦C for 20 s, and extension at 72 ◦C for 40 s; followed by denaturation at 72 ◦C for 7 min. For quantitative PCR analysis, PCR amplification was performed in a DNA Engine Opticon 1 continuous fluorescence detection system (MJ Research, Waltham, MA, USA) by using SYBR Premix Ex Taq (Takara, Japan). The total reaction volume was 10 µL containing 1 µL of cDNA/control and gene specific primers. Each PCR reaction was performed using the following conditions: 95 ◦C for 30 s, 60 ◦C for 30 s, and plate read (detection of fluorescent product) for 38 cycles followed by 7 min of extension at 72 ◦C. Melting curve analysis was performed to characterize the dsDNA product by slowly raising the temperature (0.2 ◦C/s) from 60 to 95 ◦C with fluorescence data collected at 0.2 ◦C intervals. mRNA levels of il2 and cd40l were normalized with mRNA levels of gapdh and were presented as % of controls. The fold change in gene expression was calculated using Equation (1):

f old change = 2 ∆∆CT (1) − where ∆∆CT = (CTtarget CTgapdh) at time x (CTtarget CTgapdh) at time 0. Here, time x − − − represents any time point and time 0 represents the 1 expression of the target gene in the control cells × normalized to gapdh.

4.9. IL-2 Measurement by ELISA Jurkat T cells (1 105/mL) were stimulated with immobilized anti-CD3 antibodies (20 µg/mL) × and soluble anti-CD28 (7 µg/mL) antibodies for 24 h. For conjugation, Jurkat T cells were incubated with SEE-pulsed Raji B cells for 24 h. After incubation, supernatants were collected from activated T cells or conjugation and the amount of produced IL-2 was determined by ELISA DuoSet® ELISA kit following manufacture’s instruction.

4.10. Western Blot Incubated Jurkat cells (1 106/mL) in the indicated conditions were harvested for lysis in RIPA × buffer with phosphatase inhibitor for 30 min on ice. Lysates were centrifuged at 14,000 rpm for 30 min at 4 ◦C and approximately 30 µg of the lysate was separated on 8–12% SDS–PAGE gels. Proteins were transferred on PVDF membranes (Bio-Rad, Hercules, CA, USA). Membranes were blocked in 5% skim milk for 1 h, rinsed, and incubated with indicated primary antibodies in 3% skim milk overnight. Excess primary antibodies were discarded by washing the membrane five times with TBS-T and incubated with 0.1 µg/mL peroxidase-labeled secondary antibodies (against rabbit or mouse) for 2 h. After five washes in TBS-T, bands were visualized with ECL Western blotting detection reagents (Thermo Fisher Scientific, Waltham, MA, USA) with an ImageQuant LAS 4000 (GE healthcare, Chicago, IL, USA).

4.11. Measurement of Expressions of Surface Molecules by Flow Cytometry The expressions of CD40L on T cells surface were determined by flow cytometry. Stimulated Jurkat T cells (5 105/mL) with anti-CD3 and anti-CD28 antibodies for the indicated time were harvested and × stained with anti-CD40L antibodies conjugated with APC for 30 min on ice. CD40L expressions on cells surface were acquired by flow cytometry and the expressions were shown in histogram graph. Mean fluorescence intensities were presented in line graph.

4.12. Conjugation Assay CMFDA-stained Jurkat T cells (1 105/mL) were pre-treated with 40 µM chrysophanol for 1 h. × Simultaneously, CMRA-stained Raji B cells (1 105/mL) were pulsed with 1 µg/mL SEE for 1 h. × Then Jurkat T cells and Raji B cells were mixed and incubated for 30 min or 24 h. In some cases, anti-CD40L neutralizing antibodies were added to Jurkat T cells before incubation. Conjugations were acquired by ﬂow cytometry in double-positive population. For microscopic imaging, cells were seeded Int. J. Mol. Sci. 2020, 21, 6122 12 of 13 in plate and incubated for 1 h or 24 h. Images were obtained by IncuCyte and three images were randomLy selected from each group. Conjugations between green cells (Jurkat T cells) and orange cells (Raji B cells) were counted from each selected images and percentage conjugated T cells conjugation was calculated with total T cells.

4.13. Statistics Mean values SEM were calculated from the data acquired from three independent experiments ± performed on separate days and presented in graph. One-way ANOVA were used to obtain significance (p value). * indicates differences between indicated groups considered significant at p < 0.05.

Author Contributions: Conceptualization, H.-S.L. and G.-S.J.; methodology, H.-S.L.; software, H.-S.L.; validation, H.-S.L.; formal analysis, H.-S.L.; investigation, H.-S.L. and G.-S.J.; resources, H.-S.L. and G.-S.J.; data curation, H.-S.L.; writing—original draft preparation, H.-S.L.; writing—review and editing, G.-S.J.; visualization, H.-S.L.; supervision, G.-S.J.; project administration, G.-S.J.; funding acquisition, G.-S.J. All authors have read and agreed to the published version of the manuscript. Funding: This study was funded by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-2016R1A6A1A03011325). Conﬂicts of Interest: The authors declare no conﬂict of interest.

Abbreviations

CD40L Cluster of diﬀerentiation 40 ligand ICAM-1 Intracellular adhesion molecule 1 SMAC Supramolecular activation cluster TCR T cell receptor

References

1. Xiao, B.G.; Link, H. Antigen-specific T cells in autoimmune diseases with a focus on multiple sclerosis and experimental allergic encephalomyelitis. Cell. Mol. Life Sci. 1999, 56, 5–21. [CrossRef][PubMed] 2. Lederman, S.; Yellin, M.J.; Krichevsky, A.; Belko, J.; Lee, J.J.; Chess, L. Identification of a novel surface protein on activated CD4 + T cells that induces contact-dependent b cell differentiation (Help). J. Exp. Med. 1992, 175, 1091–1101. [CrossRef][PubMed] 3. Whitmire, J.K.; Slifka, M.K.; Grewal, I.S.; Flavell, R.A.; Ahmed, R. CD40 Ligand-Deficient Mice Generate a Normal Primary Cytotoxic T-Lymphocyte Response but a Defective Humoral Response to a Viral Infection. J. Virol. 1996, 70, 8375–8381. [CrossRef][PubMed] 4. França, T.T.; Barreiros, L.A.; al-Ramadi, B.K.; Ochs, H.D.; Cabral-Marques, O.; Condino-Neto, A. CD40 ligand deficiency: Treatment strategies and novel therapeutic perspectives. Expert Rev. Clin. Immunol. 2019, 15, 529–540. [CrossRef] 5. Lederman, S.; Yellin, M.J.; Inghirami, G.; Lee, J.J.; Knowles, D.M.; Chess, L. Molecular interactions mediating T-B lymphocyte collaboration in human lymphoid follicles. Roles of T cell-B-cell-activating molecule (5c8 antigen) and CD40 in contact-dependent help. J. Immunol. 1992, 149, 3817–3826. 6. Song, G.; Zhang, Y.; Yu, S.; Lv, W.; Guan, Z.; Sun, M.; Wang, J. Chrysophanol attenuates airway inflammation and remodeling through nuclear factor-kappa B signaling pathway in asthma. Phyther. Res. 2019, 33, 2702–2713. [CrossRef] 7. Kuo, Y.H.; Lee, P.H.; Wein, Y.S. Four new compounds from the seeds of Cassia fistula. J. Nat. Prod. 2002, 65, 1165–1167. [CrossRef] 8. Coopoosamy, R.M.; Magwa, M.L. Antibacterial activity of chrysophanol isolated from Aloe excelsa (Berger). Afr. J. Biotechnol. 2006, 5, 1508–1510. 9. Tutin, F.; Clewer, H.W.B. CCXXXII.—The constituents of Cluytia similis. J. Chem. Soc. Trans. 1912, 101, 2221–2234. [CrossRef] 10. Ni, C.H.; Chen, P.Y.; Lu, H.F.; Yang, J.S.; Huang, H.Y.; Wu, S.H.; Ip, S.W.; Wu, C.T.; Chiang, S.Y.; Lin, J.G.; et al. Chrysophanol-induced necrotic-like cell death through an impaired mitochondrial ATP synthesis in Hep3B human liver cancer cells. Arch. Pharm. Res. 2012, 35, 887–895. [CrossRef] Int. J. Mol. Sci. 2020, 21, 6122 13 of 13

11. Chu, X.; Zhou, S.; Sun, R.; Wang, L.; Xing, C.; Liang, R.; Kong, Q. Chrysophanol Relieves Cognition Deficits and Neuronal Loss Through Inhibition of Inflammation in Diabetic Mice. Neurochem. Res. 2018, 43, 972–983. [CrossRef][PubMed] 12. Shen, C.Y.; Jiang, J.G.; Yang, L.; Wang, D.W.; Zhu, W. Anti-ageing active ingredients from herbs and nutraceuticals used in traditional Chinese medicine: Pharmacological mechanisms and implications for drug discovery. Br. J. Pharmacol. 2017, 174, 1395–1425. [CrossRef][PubMed] 13. Kim, S.J.; Kim, M.C.; Lee, B.J.; Park, D.H.; Hong, S.H.; Um, J.Y. Anti-inflammatory activity of chrysophanol through the suppression of NF-κB/caspase-1 activation in vitro and in vivo. Molecules 2010, 15, 6436–6451. [CrossRef][PubMed] 14. Yin, J.; Yin, Q.; Liang, B.; Mi, R.; Ai, H.; Chen, L.; Wei, X. Chrysophanol suppresses growth and metastasis of T cell acute lymphoblastic leukemia via miR-9/PD-L1 axis. Naunyn Schmiedebergs Arch. Pharmacol. 2020, 393, 273–286. [CrossRef] 15. Qian, Z.-J.; Zhang, C.; Li, Y.-X.; Je, J.-Y.; Kim, S.-K.; Jung, W.-K. Protective Effects of Emodin and Chrysophanol Isolated from Marine Fungus Aspergillus sp. on Ethanol-Induced Toxicity in HepG2/CYP2E1 Cells. Evid. Based Complement. Alternat. Med. 2011, 2011, 452621. [CrossRef] 16. Ueno, Y.; Umemori, K.; Niimi, E.-C.; Tanuma, S.-I.; Nagata, S.; Sugamata, M.; Ihara, T.; Sekijima, M.; Kawai, K.-I.; Ueno, I.; et al. Induction of apoptosis by T-2 toxin and other natural toxins in HL-60 human promyelotic leukemia cells. Nat. Toxins 1995, 3, 129–137. [CrossRef] 17. Lee, M.S.; Sohn, C.B. Anti-diabetic properties of chrysophanol and its glucoside from rhubarb rhizome. Biol. Pharm. Bull. 2008, 31, 2154–2157. [CrossRef] 18. Constant, S.L. B Lymphocytes as Antigen-Presenting Cells for CD4 T Cell Priming In Vivo 1. J. Immunol. 1999, 162, 5695–5703. 19. Boisvert, J.; Edmondson, S.; Krummel, M.F. Immunological Synapse Formation Licenses CD40-CD40L Accumulations at T-APC Contact Sites. J. Immunol. 2004, 173, 3647–3652. [CrossRef] 20. Lasserre, R.; Cuche, C.; Blecher-Gonen, R.; Libman, E.; Biquand, E.; Danckaert, A.; Yablonski, D.; Alcover, A.; Di Bartolo, V. Release of serine/threonine-phosphorylated adaptors from signaling microclusters down-regulates T cell activation. J. Cell Biol. 2011, 195, 839–853. [CrossRef] 21. Na, B.R.; Kim, H.R.; Kwon, M.S.; Lee, H.S.; Piragyte, I.; Choi, E.J.; Choi, H.K.; Han, W.C.; Lee, S.H.; Jun, C.D. Aplotaxene blocks T cell activation by modulation of protein kinase C-θ-dependent pathway. Food Chem. Toxicol. 2013, 62, 23–31. [CrossRef][PubMed] 22. Na, B.-R.; Jun, C.-D. In vitro Assessment of Immunological Synapse Formation by Flow Cytometry. Bio-Protocol. 2016, 6, e1758. [CrossRef] 23. Kvanta, A.; Gerwins, P.; van der Ploeg, I.; Nordstedt, C.; Jondal, M.; Fredholm, B.B. Stimulation of the T-cell receptors CD3 and CD2 with OKT3 and OKT11 antibodies activates a common pertussis toxin-insensitive G-protein. Eur. J. Pharmacol. Mol. Pharmacol. 1990, 189, 363–372. [CrossRef] 24. Im, N.K.; Jung, Y.S.; Choi, J.H.; Yu, M.H.; Jeong, G.S. Inhibitory effect of the leaves of Rumex crispus L. on LPS-induced nitric oxide production and the expression of iNOS and COX-2 in macrophages. Nat. Prod. Sci. 2014, 20, 51–57. 25. Ayyangar, N.R.; Bapat, D.S.; Joshi, B.S. Anthraquinones and anthrone series: Part XXXVI—A newsynthesis of chrysophanol, rhein, islandicin, emodin and physcion. J. Sci. Ind. Res. B 1961, 20, 493–497.

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).