International Immunopharmacology 26 (2015) 328–337

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International Immunopharmacology

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Immunosuppressive activity of pogostone on T cells: Blockingproliferation via S phase arrest

Ji-Yan Su a,b,1, Xia Luo b,1, Xiao-Jun Zhang b, Xiang-Liang Deng b, Zi-Ren Su b,c, Lian Zhou b, Shan-Shan Li a,Zhenhua Dai a, Yang Xu a,⁎, Xiao-Ping Lai b,c,⁎⁎a The Second Affiliated Hospital, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510006, Chinab School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, 510006, Chinac Dongguan Mathematical Engineering Academy of Chinese Medicine, Guangzhou University of Chinese Medicine, Dongguan, Guangdong, 523000, China

⁎ Corresponding author. Tel.: +86 2039318376.⁎⁎ Correspondence to: X.P. Lai, School of Chinese Materiaof Chinese Medicine, Guangzhou, P. R. China. Tel.: +86 20

E-mail addresses: yangxu@ucsd.edu (Y. Xu), lxp88@gz1 These two authors contributed equally to the paper.

http://dx.doi.org/10.1016/j.intimp.2015.04.0191567-5769/© 2015 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 2 December 2014Received in revised form 9 April 2015Accepted 10 April 2015Available online 21 April 2015

Keywords:PogostoneImmunosuppressionT cellCell cycleS phase arrest

Pogostone (PO) is one of themajor chemical constituents of the essential oil of Pogostemon cablin (Blanco) Benth.In the present study, the effect of PO on T cell responsiveness was investigated to explore its potential inimmunosuppression by a Concanavalin A (ConA)-stimulation model using splenocytes isolated from C57BL/6mice. Cytotoxicity by PO on normal splenocytes was evaluated by MTS assays. Characteristics of apoptosis,proliferation, and cell cycle were analyzed by flow cytometry. Related expressions of cyclins and cyclin-dependent kinases (CDKs) were also determined by flow cytometry. Inflammatory cytokine profiling wasperformed emplying cytometric beads assays (CBA). Moreover, the T cell-mediated delayed Type hepersensity(DTH) model was applied to evaluate the immunosuppressive activity of PO. Neither viability reduction innormal splenocytes nor apoptosis in ConA-stimulated splenocytes was observed under PO treatments.Meanwhile, PO remarkably reduced the total population of ConA-stimulated T cell, blocked T cell proliferationinduced by ConA, and inhibited the production of IFN-γ and IL-10. This blockade of stimulated T cell proliferationby POwas likely attributed to down-regulation of cyclin E, cyclin B and CDK1 and the subsequent S-phase arrest.Additionally, PO could inhibit the DTH reaction by alleviating ear swelling and inflammatory infiltrations in theDNCB-challenged ear. Taken together, PO exhibited an immunosuppressive property by directly blocking T cellproliferation as well as altering inflammatory cytokine profile, suggesting that POmay have clinical implicationsfor treating autoimmune diseases and other immune-based disorders.

© 2015 Elsevier B.V. All rights reserved.

1. Introduction

T lymphocytes are the cardinal effector cells in cell-mediatedimmunity that protect bodies from various infections and tumorgenesis[1,2]. However, activation and excessive proliferation of T cell are criticalcomponents in the pathogenesis of a multitude of inflammatorydisorders, which not only contribute to many life-threatening physicalresponses, such as allergy and transplantation rejection [3], but alsoinduce serious autoimmune diseases, including systemic lupus erythe-matosus (SLE) [4], and inflammatory bowel diseases (IBD) [5].Therefore, large amounts of endeavors have been made to find ordevelop immunosuppressant to solve these immune disturbanceproblems. Since proliferation and activation are the two prerequisites

Medica, Guangzhou University39358517.ucm.edu.cn (X.-P. Lai).

that enable T lymphocytes to drive the adaptive immunity [6,7],besides the direct cytotoxicity reagents, such as Methyprednisolone,and Azathioprine, newly clinical immunosuppressants emphasize onactivation inhibition (such as Cyclosporine), proliferation blocking(like Rapamycin), or cytokine neutralization (including Infliximab).Nevertheless, various side effects, e.g. renal toxicity, hepatotoxicity,metabolic disorders and infections, have impelled scientists andphysicians to search safer and better alternative.

T cells always stay at quiescent G0 phase when the immune systemis under normal physiological condition [8]. Upon stimulations byendogenous or exogenous antigens, T cells step into the interphaseand launch the cell cycle program, so as to make a rapid proliferationfor the purpose of immune reaction. Actually, cells can only go throughthe cell cycle when the requirements of cell cycle checkpoint would bemet, which indeed are events of the two key classes of regulatory mol-ecules, cyclins and cyclin-dependent kinases (CDKs) that determine acell’s progress through the cell cycle [9,10]. When activated by abound cyclin, CDKs perform phosphorylation that activates or inacti-vates target proteins to orchestrate coordinated entry into the nextphase of the cell cycle. CDKs are constitutively expressed in cells

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whereas cyclins are synthesized at specific stages of the cell cycle inresponse to various molecular signals. Typically, cyclin E and CDK2 areresponsible for S phase checkpoint, and cyclin B together with CDK1(cdc2) for G2/M phase.

Pogostemonis Herba is the dried aerial part of Pogostemon cablin(Blanco) Benth. In traditional Chinese Medicine, it has been widelyapplied for the treatments of emesis and sunstroke with a variety ofpharmacological activities, such as analgesia [11], immunoregulation[12,13], and anti-emesis [14]. Pogostone (C12H16O4, PO), namely 4-hydroxy-6-methyl-3-(4-methylpentanoyl)-2H-pyran-2-one (Fig. 1), isone of the major constituents of the essential oil of PogostemonisHerba. It has been reported that PO exerts potent antibacterial [15,16]and anti-fungal [17] activities. And a newly paper revealed that POpossessed the anti-inflammatory activity on a lipopolysaccharide-stimulation RAW264.7 model and a mouse endotoxic shock model[18]. In the present study, PO was investigated for its potentialinhibition of T cell responses with a Concanavalin A (ConA)-stimulationmodel in vitro and the delayed Type IV herpersensitymodel in vivo for abetter understanding.

2. Materials and methods

2.1. Animals

Male C57BL/6 mice of 18 ~ 22 g were obtained from LaboratoryAnimal Center of Guangzhou University of Chinese Medicine(Guangzhou, China) and acclimatized for 1 week before use. Micewere raised under specific pathogen-free conditions with tempera-ture of 24 ± 1 °C, humidity of 40–80% and a 12-h light/12-h darkcycle. All experiments were carried out according to the NIH Guide-lines for Care and Use of Laboratory Animals and were approvedby Bioethics Committee of Guangzhou University of ChineseMedicine.

2.2. Materials

Pogostone (PO, purity ≥ 98%) was isolated from Pogostemon cablin(Blanco) Benth as described previously [16]. In brief, dried aerial partsof P. cablin (3.4 kg) were extracted by water-steam distillation toobtained essential oil. The essential oil (11 g) was dissolved in ethylacetate (50 ml) and extracted with 4% NaOH (50 ml) for five times.The alkaline extracts were pooled and adjusted to pH2 with 10% HCl,following by the extraction with ethyl acetate for three times(200 ml). Then, the ethyl acetate extracts were pooled, washed fivetimes with distilled water (400 ml), and further evaporated to dry tountil obtain a yellow oily liquid. After crystallization from normalhexane, white PO crystals (146.2 mg, yield 0.0043%) were finallyobtained. Chemical structure of PO was identified by comparing itsspectral data (MS, 1H and 13C NMR) with those published previously[19], and the purity of PO was N98% based on high-performance liquid

Fig. 1. Structure of pogostone. The molecular weight of pogostone is 224.

chromatography (HPLC) analysis [20]. The obtained PO was tested tobe endotoxin free.

Concanavalin A type IV (ConA) and dimethyl sulfoxide (DMSO)were purchased from Sigma (USA). Carboxyfluorescein diacetatesuccinimidyl ester (CFSE) was obtained from Life Technologies-Molecular Probes (USA). Cyclophosphamide (CTX) was providedby Tonghua Maoxiang pharmaceutical Co. Ltd (Jilin, China). 2,4-Dinitrofluorobenzen was purchased fromWuhanWei Shunda technol-ogy development Co. Ltd (Hubei, China). ACK Lysis Buffer waspurchased from GibcoBRL (USA). PE-conjugated and FITC-conjugatedmonoclonal antibody against mouse CD3+, were purchased from BDPhaMingen (USA). Monoclonal antibodies against mouse cyclin B andCDK2 were from Cell Signaling Technology (USA), cyclin E fromThermoFisher Scientific (USA), and CDK1 (cdc2) from Abcam (USA).Alexa fluor 647-conjugated anti-mouse IgG (H + L), anti-rabbit IgG(H + L) and the relevant isotype were from Cell Signaling Technology(USA). CellTiter 96® AQueous One Solution Cell Proliferation Assay(MTS) was from Promega (USA). Apoptosis Assay Kit and Cell CycleAssay Kit were purchased from Lianke Biology Inc (Hangzhou, China).Mouse Inflammation Cytometric Bead Array (CBA) Kit was purchasedfrom Becton Dickenson (USA).

2.3. Cell preparation and ConA stimulation

Splenocytes were isolated as described previously with minormodification [21]. Briefly, C57BL/6micewere sacrificed, and the spleenswere aseptically removed and ground in pre-cold PBS by passingthrough a 70 μm strainer. After centrifuge at 300 × g for 5 min at 4 °C,erythrocytes were lysed in ACK Lysis Buffer for 5 min at room tempera-ture. Cells were collected by centrifugation at 300 × g for 5 min at 4 °C,and washed twice with pre-cold PBS. Then cells were resuspended incomplete RPMI1640 containing 10% heat-inactivated FBS, and 100 IU/mL of penicillin and streptomycin except experiments of proliferationassay. For proliferation assay, the isolated splenocytes were resuspend-ed in 5%FBS/PBS, counted and incubatedwith 2.5 μMCFSE at a density of5 × 106 cells/mL for 8 min in dark at room temperature, washed twicewith 5% FBS/PBS, resuspended in complete medium. Cell counts wereperformed on a hemocytometer and cell viability was determined bytrypan-blue dye exclusion, and in all cases cell viability was higherthan 95%. The culture media were maintained in a humidifiedatmosphere of 5% CO2 at 37 °C.

For ConA stimulation, 500 μL of splenocyteswere seeded at a densityof 3 × 106 cells/mL onto 24-well plates (triplicate wells), and pre-treated with 500 μL of PO (20, 40, 80 μM) for 4 h following by astimulation of ConA (1 μg/mL) for another 72 h.

2.4. Cytotoxicity assay

100 μL of splenocytes were seeded at a density of 3 × 106 cells/mL in96-well plates (sextuplicate wells in each group), and treated with100 μL PO in complete RPMI 1640 medium of multiple concentrations(0, 5, 10, 20, 40, 80 μM, with no more than 0.1% DMSO). After 44 h or68 h, 40 μL of MTS was added to each well and the cells were furtherincubated for 4 h at 37 °C with 5% CO2. The optical density wasmeasured at 490 nm on a microplate reader.

2.5. Apoptosis analysis

Apoptosis was analyzed by Annexin V-FITC and Propidium Iodide(PI) staining according to the manufacture’s instruction. Briefly, cellswere harvested, andwashed oncewith PBS. Then cells were resuspend-ed in 500 μL of binding buffer and stained with Annexin V-FITC and PIfor 5 min in dark at room temperature. Characteristics of the cellswere acquired and analysed on a FC500 flow cytometer (Beckman,USA), using CXP software (Beckman).

Fig. 2. Viability of splenotye after treatment with PO for 48 h (A) and 72 h (B). Values were presented as means ± SD (n = 3).*p b 0.05 and **p b 0.01 versus control group.

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2.6. Proliferation assay

CFSE-labeled cells were stainedwith PE-conjugated anti-CD3 for 30-min on ice, washed in PBS with 1% BSA twice, and resuspended in 2%paraformaldehyde. Percentage of the target cells and divided ratiowere determined from the CFSE profiles on a FC500 flow cytometer(Beckman, USA), using CXP software (Beckman).

2.7. Cell cycle assay

Cells were stained with FITC-conjugated anti-CD3 for 30 min on ice,washed in PBS with 1% BSA. The cells were fixed and permeabilized bycold 70% ethanol (−20 °C) containing 10% FBS for 30 min. After twocold PBS washes, cells were labeled by PI staining solution (50 g/mL PIand 0.1% Triton X-100 in PBS) in the dark for 10 min at room tempera-ture before analysis with flow cytometry. Cell cycle propagation, as

Fig. 3.Apoptosis analysis of the ConA-stimulated splenocyte. (A) Typical FCMpictures from eachpresented as means ± SD (n = 3). #p b 0.05 and ##p b 0.01 versus control group, * p b 0.05 a

determined byDNAploidy,was assessed on the basis of PI incorporationusing Modfit software.

2.8. Intracellular cytokine expression assay

Cells were stained with FITC conjugated anti-CD3 for 30 min on ice,washed in PBS with 1% BSA, and then resuspended in 4% formaldehydeat 37 °C for 10min followingby an incubation in ice for 1min. Cellswerewashed with 1%BSA/PBS and incubated with FACS Perm/Wash Solution(BD Biosciences) for 15 min in the dark, washed with Perm/WashSolution, and then stained with primary antibodies against Cyclin E,Cyclin B, p21, CDK1 and CDK2 on ice for 1 h. Cells were washed andstained with Alexa fluor 647-conjugated secondary body for 30 min.At last, cells were washed again and resuspend in PBS. Matchedfluorochrome-conjugated isotype control monoclonal antibodies wereused to establish gating. Characteristics of the cells were acquired and

group. (B) All datawere statistically analyzed anddisplayed in column charts. Valueswerend **p b 0.01 versus ConA group.

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analysed on a FC500 flow cytometer (Beckman, USA), using CXPsoftware (Beckman).

2.9. Determination of inflammation cytokine profiles

Tomeasure the release of cytokines, including Interleukine-6 (IL-6),Interleukine-10 (IL-10), Monocyte chemotactic protein (MCP-1),Interferon-γ (IFN-γ), and Tumor nerosis factor (TNF), the commercialBecton Dickenson Mouse Inflammation Cytometric Bead Array (CBA)Kit was applied. The assays were performed according to manufac-turer’s instruction on supernatant collected 72 h after the Con A-stimulation in the presence or absence of PO. For the Mouse Inflamma-tion CBA kit, 25 μL of supernatant was stained with the mixture ofMouse Inflammation capture bead suspension and the PE detectionreagent. After 2 h of incubation, samples were washed and then

Fig. 4. Proliferaion analysis of the ConA-stimulated splenocyte. Typical pictures from each groupresponse to Con A with or without PO treatment for 72 h. (C, D) All data were statistically an#p b 0.05 and ##p b 0.01 versus control group, * p b 0.05 and **p b 0.01 versus ConA group.

analyzed byusing the BDCBA soft-ware.Mouse Inflammation standardsprovidedwith the kit were appropriately diluted and used in parallel tosamples for preparation of the standard curves.

2.10. DNCB-induced T-cell-mediated delayed type hypersensitivity (DTH)reaction

Male C57BL/c mice were randomly divided into six groups, six ineach. Mice were initially sensitized with 50 μL of 1% DNCB dissolved inacetone on the shaved abdominal skin of recipients. DTH reaction waselicited 6 days later by smearing 10 μL of 1% DNCB on both sides of theleft ear. On the first day of immunization, the immunized mice wereorally administered with PO at the doses of 10, 20 or 40 mg/kg(dissolved in 2% polysorbate 80) daily for 7 consecutive days. Thepositive control group was given a gavage of cyclophosphamide (CTX)

showing CD3+ T cell proportion (A) and proliferating percentage (B) for CFSE staining inalyzed and displayed in column charts. Values were presented as means ± SD (n = 3).

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at a dose of 37 mg/kg for 7 days once daily. The normal and modelgroups received the same volume of 2% polysorbate 80. 24 h after thesecond challenge, DTH reaction was measured as indicated by the in-crease in the ear patch weight (8-mm punches) between the left andright ear. All the mouse left ears were removed and fixed in 4% formal-dehyde (pH 7.2) for histological assessment of the lesions by hematox-ylin and eosin (HE) staining.

2.11. Statistical analysis

A minimum of 10000 cells were assessed in FCM analysis. Allexperiments in vitrowere performed in at least triplicate, and the resultsare expressed as means ± SD. Statistical analysis was performed byone-way analysis of variance (ANOVA) using SPSS 18.0. A post–hocLSD test was applied to analyze the difference among the groupsunder homogeneity of variance, if not, a Dunnett’s test would beapplied. #p b 0.05 and ##p b 0.01 as compared to control or normalgroup; *p b 0.05 and **p b 0.01 as compared to ConA group or modelgroup.

3. Results

3.1. PO had no toxicity on splenocyte

As shown in Fig. 2, viability of splenocyte was not affected after a48 h- or 72 h-incubation with 5 to 80 μMPO compared with the controlgroup, indicating that PO had no toxicity on splenocytes (Fig. 2).

3.2. PO inhibited ConA-stimulated T cell proliferation withoutinducing apoptosis

By Annexin V-PI staining, it was found that no more apoptosis wasobserved in the ConA-stimulated splenocyte treated by 20 μM and40 μMPO, while 80 μMPO even held back the apoptosis as demonstrat-ed by that fewer cells were stained with Annexin V (Fig. 3, p b 0.01).To study the proliferation characteristics after treatment, T cells werefigured out by PE-conjugated anti-CD3 from the total splenocytes.After ConA stimulation, T cells proliferated significantly as indicated

Fig. 5. Cell cycle analysis of the ConA-stimulated splenocyte. (A) The cell cycle distribution ostatistically analyzed and displayed in column charts. Values were presented as means ± SDConA group.

by the increased percentages of total and dividing T cell (Fig. 4,p b 0.01). By contrast, PO evidently decreased the percentage of thetotal T cells, at the same time reduced the proliferation proportion oftotal T cells (Fig. 4, p b 0.01). And we also found that PO could signifi-cantly inhibit the proliferations of both subsets of T cell, the CD4+ andCD8+ T cells (data was shown in Supplement). All these suggestedthat PO could inhibit ConA-stimulated T cell proliferation directly, butnot via the way of apoptosis.

3.3. PO induced an S phase arrest in T cells upon ConA sitmulation

As has been revealed that PO inhibited the ConA-mediated T cellsproliferation directly, we analyzed the impact of PO on cell cycle toreveal more details about the immunosuppressive activity of PO. AfterConA stimulation, more T cells stepped into S phase and G2/M phase,while PO (20, 40 and 80 μM) led to an obvious increase in the numberof cells in the S phase with an evidently decreased percentage of cellsin the G2/M phase, suggesting that PO would arrest T cells in S phaseupon ConA stimulation (Fig. 5, p b 0.01).

3.4. S phase arrest by PO was associated with cyclin and CDK reduction

Progression through the cell cycle involves accumulation, sequentialassembly and activation of cyclins and CDKs. To elucidate how PO arrestT cells in S phase upon stimulation, the S phase- andG2/Mphase-relatedcyclins and CDKs were analyzed by intracellular staining FCM. Resultsshowed that percentages of CDK2-expressing were decreasedsignificantly after ConA stimulation (Fig. 6, p b 0.01), while that of cyclinE-expressing T cell increased (Fig. 7, p b 0.01). By PO treatment, theproportion of cyclin E-expressing T cells was reduced, of which the80 μM PO group showed an evident decrease (Fig. 7, p b0.05), whilethat of CDK2-expressingwas not affected (Fig. 6), suggesting that cyclinE expression was down-regulated by PO. For G2/M phase, ConAchallenge induced an evident increase on cyclin B-expressing andCDK1-expressing cells (Figs. 8, 9). By contrast, PO remarkably loweredthe T cell population of cyclin B and CDK1 in a dose-dependentmanner,quite consistent with the G2/M phase decrease by PO, suggesting adown regulation on the expressions of cyclin B and CDK1 (Figs. 8, 9).

f T cell was determined using flow cytometry and analyzed by Modfit. (B) All data were(n = 3). #p b 0.05 and ##p b 0.01 versus control group, * p b 0.05 and **p b 0.01 versus

Fig. 6. CDK2 expression analysis of the ConA-stimulated T cell. (A) Typical FCM pictures from each group. (B) All data were statistically analyzed and displayed in column charts. Valueswere presented as means ± SD (n = 3). #p b 0.05 and ##p b 0.01 versus control group, * p b 0.05 and **p b 0.01 versus ConA group.

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3.5. Impact of PO on the inflammation cytokine profile

As showed in Fig. 10, levels of all the five cytokines (IL-6, IL-10,MCP-1, IFN-γ, and TNF) soared significantly after ConA stimulation for 72 h(p b 0.01). PO exhibited various impacts on different cytokines. Uponthe stimulation, PO inhibited the releases of pro-inflammatory IFN-γand anti-inflammatory IL-10 obviously, while those of IL-6 or TNFwere not affected (p b 0.01). However, the secretion ofMCP-1 displayeda trend of increase after PO treatment when compared with that ofConA challenge, and 20 μM PO even made a significant increase onMCP-1 (p b 0.01).

Fig. 7. Cyclin E expression analysis of the ConA-stimulated T cell. (A) Typical FCMpictures fromwere presented as means ± SD (n = 3). #p b 0.05 and ##p b 0.01 versus control group, *p b 0

3.6. Effect of PO on DNCB-induced DTH response in C57BL/c mice

The immunosuppressive activity of PO was test on the DNCB-induced DTH model. As shown in Fig. 11A, the ear swelled significantlyin Model group compared with control group (p b 0.01). CTX and PO atdoses of 10, 20 or 40 mg/Kg significantly reduced the ear swelling inDNCB-induced mice compared with that of Model group (p b 0.01),and PO at 40 mg/Kg exhibited the strongest immunosuppressive effectagainst DTH reaction.

HE staining provided more details about the effect of PO. Comparedwith that of control group, DNCB challenge caused evident tissue

each group. (B) All data were statistically analyzed and displayed in column charts. Values.05 and **p b 0.01 versus ConA group.

Fig. 8. CDK1 expression analysis of the ConA-stimulated T cell. (A) Typical FCM pictures from each group. (B) All data were statistically analyzed and displayed in column charts. Valueswere presented as means ± SD (n = 3). #p b 0.05 and ##p b 0.01 versus control group, *p b 0.05 and **p b 0.01 versus ConA group.

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hypertrophy and edema (Fig. 11B b), which was manifested by theremarkably increased monocytes, eosinophils and neutrophils withinfiltration into perivascular connective tissue. By contrast, CTX admin-istration remarkably ameliorated tissue swellings, and inflammatorycell infiltration was decreased (Fig. 11B c). PO could also inhibit thiscellular immune response by relieving tissue edema, as well as theinflammatory infiltration (Fig. 11B d, e, f).

4. Discussion

Upon recognition of their specific antigen, lymphocytes, including Tlymphocyte and B lymphocyte, are capable of massive clonal expansion

Fig. 9. Cyclin B expression analysis of the ConA-stimulated T cell. (A) Typical FCMpictures fromwere presented as means ± SD (n = 3). #p b 0.05 and ##p b 0.01 versus control group, *p b 0

[22]. That is, T cells have to proliferate for the purpose of launching theadaptive immune response against endogenous or exogenous stimula-tion. Since it has become evident that corticosteroid was associatedwith significant morbidity to the patient, immunosuppressants thatare able to perturb the lymphocyte proliferation are sought to sparecorticosteroid in the clinic of inflammation controlling. In the presentstudy, immunosupprression potential of PO was investigated from theaspects of proliferation blocking.

As demonstrated in our data, neither viability reduction norapoptosis upon ConA stimulation were observed in splenocytesafter PO treatment, even in which the high dose of PO held back the ap-optosis caused by ConA. Meanwhile, PO remarkably reduced the total

each group. (B) All data were statistically analyzed and displayed in column charts. Values.05 and **p b 0.01 versus ConA group.

Fig. 10. Cytokine profile analysis of the ConA-stimulated splenocytes. All data were statistically analyzed and displayed in column charts. Values were presented as means ± SD (n= 3).#p b 0.05 and ##p b 0.01 versus control group, *p b 0.05 and **p b 0.01 versus ConA group.

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population and proliferation percentage of the ConA-stimulated T cell,as well as those of CD4+ and CD8+ subsets. ConA is a polyclonalphytolectin that non-specifically stimulates and activates T cell toproliferate following by the activation-induced cell death (AICD). Apo-ptosis is the requirement to maintain tissue homeostasis, functioningon eliminating the redundant, damaged, or infected cells [23,24]. There-fore it could be observed that there existed apoptosis in splenocytesunder ConA stimulation. Since T cells would stay rest and maintain aconstant population either in vivo or in vitro unless being stimulated,and decreased cell population is believed resulting from necrosis [25],apoptosis [26] or proliferation blocking, it can be concluded that byruling out the cause of necrosis or apoptosis, the growth inhibition byPO was a direct consequence of proliferation blocking, moreover to re-verse the AICD induced by ConA. On the other hand, going through acomplete cell cycle is the necessity for cell proliferation and division,while arrest on any phase of the cell cycle would definitely result in aproliferation blocking. Hence, it can be hypothesized that PO inducean arrest on cell cycle as it has been proved that the inhibition of POon T cell proliferation was irrelevant to cytotoxicity or to apoptosis. Ashas been expected, data from the present study proved that POsignificantly increased the T cell population in S phase accompanying

with a reduction in G2/M phase, indicating that the proliferationblocking of PO was closely related to an S phase arrest.

To further analyze the mechanism underlying the S phase arrest byPO, cyclin and CDKs expressions in T cells were determined by FCM,mainly focusing on the G1/S phase and G2/M phase checkpoints due tothe dramatic changes being observed in these two phases. Cell cyclecheckpoints, the molecular events for cell cycle regulation, involveprocesses crucial to the survival of a cell, for example the detectionand repair of genetic damage as well as the prevention of uncontrolledcell division [9]. Cells cannot proceed to the next phase until checkpointrequirements have been met, that is the cylcin-CDK complex must beformed and activated. There are twomain checkpoints: the G1/S check-point and the G2/M checkpoint. For G1/S checkpoint, the transientproduced cylcin Ewould bind to CDK2, then the cyclin E-CDK2 complexwas formed to push the cell from G1 to S phase by participating in therelease of the transcription factor E2F and phosphorlating theprereplication complex [27]. For G2/M phase, activation of the cyclinB-CDK1 complex (the M phase promoting factor, MPF) induces break-down of nuclear envelope and initiation of prophase, and subsequently,its deactivation causes the cell to exit mitosis [28], but the activity ofcyclin B-CDK1 complex would be suppressed due to the imposition of

Fig. 11. Effects of PO on DNFB-induced DTH reaction in C57BL/6 mice. (A) The ear weightchange (mg) was calculated as the difference between the weights of untreated andDNCB-treated ear punches 24 h after challenge. (B) Typical histology pictures of theDNCB-elicitated left ear (400×). (a) Control group, (b) Model group, (c) CTX group, (d,e, f) PO 10, 20, 40 mg/Kg groups. The Values were presented as means ± S.D. (n = 6).#p b 0.05 and ##p b 0.01 versus Normal group, *p b 0.05 and **p b 0.01 versusModel group.

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inhibitory phosphorylations on the CDK1 kinase as well as down-regulation of cyclin B levels [28]. Our data showed that PO maderemarkable reductions on cyclin E, cyclin B and CDK1, while thepopulation of S phase increased significantly accompanied with a sub-stantial decrease in G2/M phase after PO treatment. Because cyclin E isexpressed precisely when needed and then is rapidly degraded in thelater S phase [29], meanwhile down-regulation of cyclin B or CDK1levels would hold up the G2/M checkpoint [28], it can be speculatedthat although the ConA-stimulated T cells had stepped into the later Sphase when cyclin E had degraded, PO induced a failure of G2/M check-point in T cells by down-regulating the expressions of cyclin B andCDK1, thereby stalling the ConA-caused proliferating in the later S

phase. In other words, PO led to an S phase arrest to block T cell prolif-eration upon the stimulation of ConA.

In addition to proliferation inhibition, affects of PO on the immuneresponse was also investigated by analyzing the cytokine profile fromthe ConA-stimulated splenocyte. Basically, immune responses arecomprehensive actions by several cells under the drive of cytokines,which are important glycoproteins used for signaling between cells toaffect the behavior of cells, including IFNs, ILs, lymphokines,chemokines, and TNFs. Specifically, the pro-/anti- inflammatory cyto-kine balance plays a key role in the initiation and regulation of the im-mune response [30,31]. IFNs belong to the large class of Th1-derivedcytokine, known as to trigger protective defenses of the immune systemthat help eradicate pathogens. But in response to certain cases, e.g. theinflammatory bowel disease, IFNs (especially the IFN-γ) contribute tothe progression by inducing production of certain chemokines impor-tant for inflammatory cell recruitment [32]. IL-6, IL-10 are interleukinsthat mainly synthesized by CD4+ T lymphocytes with various impacton the immune system. As a crucial pro-inflammatory cytokine, IL-6 in-duces pleiotropic influence on antigen-specific immune responses andinflammatory reactions, especially response to infection and tissueinjuries [33,34]. IL-10 is a potent anti-inflammatory cytokine thatinhibits the synthesis of a number of cytokines, including IFN-γ, IL-2,IL-3, TNF, and GM-CSF produced by activated macrophages or helperT cells, thus counteracting the hyperactive immune response to pro-tect the body from excessive cell and organ damage [35]. MCP-1 is asmall CC chemokine, capable of recruiting monocytes, memory Tcells, and dendritic cells to the sites of inflammation produced byeither tissue injury or infection [36,37]. TNF-α is a major pro-inflammatory cytokine involved in systemic inflammation andstimulate the acute phase reaction. It is mainly produced by activat-ed macrophages, although it can be produced by many other celltypes such as T cells, NK cells, neutrophils, mast cells, eosinophils,and neurons. As shown in the present data, releases of the cytokines(IL-6, IL-10, MCP-1, IFN-γ, and TNF) from the normal splenocytewere dramatically increased upon the T cell-specific mitogen stimu-lation of ConA in the wake of T cell activation and proliferation. Bycontrast, PO remarkably suppressed the secretions of IFN and IL-10despite that no changes were observed in IL-6, MCP-1, or TNF.These cytokine profile changes suggested that, as a consequence ofthe S phase arrest, PO inhibited the T cells proliferation and thefollowing production of releases of the Th1-derived IFN-γ and Th2-derived IL-10, thus posing a comprehensive suppression on T cells.

To confirm the immunosuppression activity of PO in vivo, DTHresponse was employed to test whether PO would be effective againstT-cell-mediated immune response. DTH response is an inflammationcharacterized by mononuclear infiltration and tissue damage causedby stimulated CD4+ T helper cells. Clinically, DTH develops in individualwho was previously sensitized with a contact allergen, mainly infec-tions or hapten, and then was re-exposed to the same or a structurallyclosely related allergen. Results showed that PO at dosages of 10, 20,40 mg/kg could significantly inhibit this cellular immune response, asindicated by alleviating ear swelling and by relieving the infiltrationsof monocytes, eosinophils and neutrophils in the challenged ear. Ashas revealed in the in vitro experiment, the inhibition against DTH byPO would be a reasonable consequence of its immunosuppression onT cells, through proliferation blocking and cytokines inhibition.

Taken together, it is, for the first time, revealed that PO exhibited animmunosupprression potential by directly blocking T cell proliferationconcomitant with suppression on the inflammatory cytokine profile,which was further confirmed by its inhibition against DTH reaction.Moreover, data from the present work suggested that the immunosup-pressive activity of PO would be a bluntness on the immune responserather than a destruction on the adaptive immune system, as has beenmanifested by the fact that PO would direct inhibit T cell proliferationwithout causing necrosis nor apoptosis on T cells. It has been wellknown that rapamycin is one of the popular immunosuppressants

337J.-Y. Su et al. / International Immunopharmacology 26 (2015) 328–337

that inhibit lymphocyte proliferation by interfering their metabolismvia PI3K signaling pathway, a crucial target in the immunotherapyfor its importance in cell proliferation and metabolism [38]. It is gettingmore widely used in clinic for prevention of renal transplantationrejection since 1999 [39] because it could minimize both rejection andcomplication of immunosuppression such as infections and nephrotox-icity [38]. Therefore, the possible regulation of PO on the PI3K signalingpathwaywould be the following task for the research extension of PO. Itis also important to understand how PO interferes the differentiationand polarization of T cell under the excessive immune response.

5. Conflicts of interest

The authors declare that there were no conflicts of interest.

Acknowledgements

This study was financially supported by grants from the GuangdongProvince Universities and Colleges Pearl River Scholar Funded Scheme(2011), the Science and Technology Project for Medical and HealthInstitution of Dongguan (Grant No. 2012105102009), the National KeyTechnology Support Program (Grant No.2012BAI29B00), the Special-ized Research Fund for the Doctoral Program of Higher Education(Grant No. 20134425110009), the National Natural Science Foundationof China (Grant No.81173534, and No.81303200), the Science andTechnology Cooperation Project of Hongkong, Macaw and Taiwan(Grant No. 2014DFH30010), the Special Funds from Central Finance ofChina in Support of the Development of Local Colleges and University[Educational finance Grant No.276(2014)].

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.intimp.2015.04.019.

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