International Immunopharmacology 20 (2014) 307–315

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International Immunopharmacology

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Cholecystokinin octapeptide regulates the differentiation and effectorcytokine production of CD4+ T cells in vitro

Jing-Ge Zhang a, Jun-Xu Liu b, Xian-Xian Jia c, Jing Geng c, Feng Yu c, Bin Cong c,⁎a Department of Pathophysiology, Institute of Basic Medicine, Hebei Medical University, Shijiazhuang, Chinab Department of Laboratory Animal Science, Hebei Medical University, Hebei Key Lab of Laboratory Animal Science, Shijiazhuang, Chinac Hebei Key Laboratory of Forensic Medicine, Hebei Medical University, Shijiazhuang, China

⁎ Corresponding author at: Hebei Key Laboratory of FoUniversity, 361 Zhongshan East Road, Shijiazhuang, Hebe86265645; fax: +86 311 86265607.

E-mail addresses: zhangjg001@163.com (J.-G. Zhang),pigejxx@qq.com (X.-X. Jia), 46232839@qq.com (J. Geng),hbydbincong@126.com (B. Cong).

http://dx.doi.org/10.1016/j.intimp.2014.03.0131567-5769/© 2014 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 28 November 2013Received in revised form 19 March 2014Accepted 19 March 2014Available online 2 April 2014

Keywords:CholecystokininTranscription factorsSignature cytokinesT helper cells

Cholecystokinin octapeptide (CCK-8), an immunomodulatory peptide, can promote or suppress the develop-ment or function of specific CD4+ T cell subsets by regulating antigen-presenting cell functions. In the currentstudy, we investigatedwhether CCK-8 exerts a direct effect on T cells through influencing differentiation and cy-tokine production of distinct CD4+ T cell subsets in vitro. Our results showed that CCK-8 differentially affects thedevelopment and function of CD4+ T cell populations, with a negative influence on Th1 and Th17 cells and pos-itive regulatory effect on inducible T regulatory cells (iTreg). Notably, CCK-8 suppressed Th1 while slightly en-hancing Th2 development and cytokine production. Similarly, CCK-8 inhibited the differentiation of Th17 cellsand promoted Foxp3 expression. L-364,718 and LY-288,513, selective antagonists of CCK1R and CCK2R, respec-tively, suppressed the effects of CCK-8 on CD4+ T cell subset-specific transcription factors. Our findings stronglyindicate that CCK-8 exerts a direct effect on T cells, which is dependent on CCKRs, particularly CCK2R. The collec-tive results aid in further clarifying themechanism underlying the anti-inflammatory and immunoregulatory ef-fects of CCK-8.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

CD4+ T cells play critical roles in mediating adaptive immunity to avariety of pathogens [1]. After encountering antigen, naive CD4+ T cellsdifferentiate into discrete effector T cells, including, but not limited to,Th1, Th2, Th17 and inducible T regulatory (iTreg) cell subsets thatproduce distinct patterns of cytokines with different functions in theimmune system. Th1 cells that produce IFN-γ and express the transcrip-tion factor T-box 21 (T-bet) are crucial for the clearance of intracellularpathogens [2,3]. Th2 cells secreting IL-4, IL-5, and IL-13 and expressingthe transcription factor GATA-binding protein 3 (GATA-3) play a criticalrole in protection against extracellular pathogens and induction of aller-gic responses [4]. Th17 cells, known to produce the proinflammatory cy-tokine IL-17 and express the transcription factor retinoic acid orphanreceptor gamma (RORγt), are related to tissue inflammation and exac-erbation of autoimmune pathology [5–12]. iTreg cells, together withnaturally occurring T regulatory (nTreg) cells, express the transcriptionfactor Foxp3, and control the inflammatory and immune responses tosuppress the development of autoimmune diseases [13,14]. CD4+ T

rensic Medicine, Hebei Medicali 050017, China. Tel.: +86 311

liujx6688@sina.com (J.-X. Liu),fengyu@hebmu.edu.cn (F. Yu),

cells producing distinct cytokine profiles develop during immune re-sponses to invading pathogens. An appropriate response facilitatespathogen elimination, while an incorrect or limited response can resultin chronic infection [15]. Emerging evidence suggests that naive CD4+ Tor partially differentiated cells possess sufficient plasticity to convertinto other types of effector cells under specific conditions [16–19].Therefore, impacts on differentiation into Th1, Th2, Th17 and Tregcells may represent an underlying basis of the proinflammatory oranti-inflammatory and immunoregulatory actions of pharmacologicalagents.

Cholecystokinin (CCK) is a brain–gut peptide, with sulfated chole-cystokinin octapeptide (CCK-8) as the biologically predominant and ac-tive form. CCK exerts pleiotropic functions in several systems, includingthe immune system, through activation of specific receptors (CCKRs)[20]. Administration of CCK-8 suppresses the expression of proinflam-matory genes, such as intracellular adhesion molecule-1 (ICAM-1), CCchemokine ligand 2 and tumor necrosis factor-α (TNF-α), and infiltra-tion of macrophages in diabetic kidneys. Furthermore, CCK-8 has beenshown to inhibit both TNF-α expression and chemotaxis in culturedTHP-1 cells [21]. The group of Luyer [22] reported that high-fat enteralnutrition stimulates CCKRs, leading to inhibition of the inflammatory re-sponse, whereas CCK antagonists significantly enhance release of theproinflammatory cytokines, TNF-α and interleukin (IL)-6. McDermottand co-workers [23] also demonstrated a functional connection be-tweenCCK, inflammation andCD4+T lymphocytes. Earlier experiments

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by our group indicated that CCK-8 exerts several immunomodulatoryeffects. Specifically, CCK-8 inhibited phenotypic and functional matura-tion and downregulated CD80 and CD86 expression in dendritic cells(DCs), suppressed costimulatory activity and immunoglobin G1 inlipopolysaccharide (LPS)-activated B cells, and decreased secretion ofproinflammatory cytokines, such as TNF-α, IL-1β and IL-6, while in-creasing production of anti-inflammatory cytokines, such as IL-4, inLPS-activated macrophages and B cells [24–26]. CCK-8 additionallymodulated the Th1/Th2 imbalance in keyhole limpet hemocyanin(KLH)-immunized mice and significantly suppressed the incidenceand severity of collagen-induced arthritis (CIA) in mice through inhibi-tion of Th17 polarization mediated by DCs [27,28]. These results clearlyindicate that CCK regulates the inflammatory and immune response.Moreover, CCK-8 partially exerts its effects by modulating antigen-presenting cell (APC) functions. The issue of whether CCK-8 exerts a di-rect effect on T cells through influencing the development of CD4+ Tsubsets has not been established to date.

In the current study, we activated and differentiated purified CD4+ Tcells from mice spleens under Th1-, Th2-, Th17- and iTreg-polarizingconditions in vitro. The effects of CCK-8 on differentiation and effectorcytokine production of Th1, Th2, Th17 and iTreg cells were compared.

2. Materials and methods

2.1. Animals

C57BL/6 mice, 8 to 12 weeks old, were obtained from LaboratoryAnimal Center of Hebei Province (Shijiazhuang, China).

2.2. Reagents

A mouse CD4+ T cell isolation kit was purchased from MiltenyiBiotec (Bergisch Gladbach, Germany). LEAF™-purified anti-CD3ε(145-2C11) and LEAF™-purified anti-CD28 (35.71) were obtainedfrom Biolegend (San Diego, CA). Recombinant mouse IL-12, IL-2, IL-4,IL-6 and recombinant human TGF-β were from PeproTech (Rocky Hill,NJ). Antibodies, including anti-IL-4, anti-IFN-γ, FITC-anti-CD4 (GK1.5),PE-anti-CD4, FITC-anti-IFN-γ, PE-anti-IL-4, PE-anti-CD25 (PC61), PE-anti-IL-17 (TC11-18H10), mouse regulatory T cell staining kit (FJK-16 s) andmonensin solution (1000×)were purchased from eBioscience(SanDiego, CA). PMA (phorbol 12-myristate 13-acetate) and ionomycinwere acquired from Alexis (Farmingdale, NY). Sulfated CCK-8 was pur-chased from SigmaChemical Co. (St. Louis,MO, USA). L-364,718 and LY-288,513 (selective antagonists of CCK1R and CCK2R, respectively) wereobtained from Tocris Bioscience (Ellisville, MO, USA), anti-CCK1R andanti-CCK2R from Santa Cruz (CA, USA), mouse IFN-γ and IL-4 ELISAkits from Neobioscience (Beijing, China), and mouse IL-10 and IL-17ELISA kits from Bender (Burlingame, CA).

2.3. Cell isolation

CD4+ T cells were isolated from mice splenocytes with mouse anti-CD4 (L3T4) magnetic microbeads and LS column (Miltenyi Biotec,Auburn, CA), followed by MACS selection according to themanufacturer's protocol. The purity of sorted cells was detected usingflow cytometry (N95% for CD4+ cells).

2.4. In vitro T cell differentiation

Purified CD4+ T cells were resuspended at a concentration of1 × 106 cells/well in RPMI 1640 (Invitrogen, Carlsbad, CA) supple-mented with 10% fetal calf serum, 2 mM glutamine, 100 IU/ml pen-icillin and 0.1 mg/ml streptomycin, and cultured in 24-well plates inthe presence of plate-bound anti-CD3ε (5 μg/ml) and soluble anti-CD28 (2 μg/ml). IL-12 (5 ng/ml) and anti-IL-4 (10 μg/ml) wereadded for Th1 differentiation, and IL-4 (10 ng/ml) and anti-IFN-γ

(10 μg/ml) for Th2 differentiation. TGF-β (1 ng/ml), IL-6 (20 ng/ml),anti-IL-4 (5 mg/ml) and anti-IFN-γ (10 μg/ml) were added for Th-17 differentiation, and TGF-β (5 ng/ml) and anti-IL-4 (5 mg/ml) foriTreg differentiation. Cultures were additionally supplemented withIL-2 (50 U/ml) on day 2. Cells were cultured for 4 days in the ab-sence or presence of CCK-8 (10−10 mol/l to 10−6 mol/l).

2.5. Flow cytometry analysis

Cells previously stimulated with plate-bound anti-CD3ε and solubleanti-CD28 for 4 days were re-stimulated with PMA (50 ng/ml),ionomycin (1 mg/ml) and monensin (1 μl per 1.0 ml) for 5 h at 37 °Cin 5% CO2. Next, cells were fixed and permeabilized using Cytofix/Cytoperm, and stained with single fluorochrome-conjugated anti-CD4,anti-IFN-γ, anti-IL-4, anti-IL-17, anti-CD25 and anti-Foxp3 antibodies,according to the manufacturer's protocol. Cells were subsequently ana-lyzed using a FACSCalibur with CellQuest software (BD Biosciences,Mountain View, CA).

For CCK1R and CCK2R, CD4+ T cells were incubatedwith anti-CCK1Rand anti-CCK2R or isotype control Abs for 30min at 4 °C. After a secondincubation with the appropriate anti-isotype FITC-conjugated Abs for30 min at 4 °C, cells were washed and analyzed using a FACSCaliburcytofluorimeter with CellQuest software (BD Biosciences).

2.6. Quantitative and semiquantitative PCR

Quantitative PCR was applied for detection of the mRNA levels oftranscription factors (T-bet, Gata-3, RORγt and Foxp3) and cytokines(IFN-γ, IL-4, IL-17 and IL-10). Total RNA was extracted from culturedCD4+ T cells using TRIzol Reagent (Invitrogen, Carlsbad, CA), followedby quantitation with the NanoDrop spectrophotometer (NanoDropTechnologies, Wilmington, DE), and the aliquots (0.5 μg) subjected toreverse transcription according to the manufacturer's protocol. One-tenth of the RT reaction was used for real-time PCR with Power SYBRGreen PCR Master Mix (Applied Biosystems, Warrington, UK) and theABI-PRISM 7500 Sequence Detector System (Applied Biosystems). Theprimers used for amplification were as follows: T-bet sense, 5′-ACCAGAGCGGCAAGTGGG-3′ and antisense, 5′-TGGACATATAAGCGGTTCCCTG-3′; GATA3 sense, 5′-CTGGAGGAGGAACGCTAATG-3′ and antisense,5′-AGAGATGTGGCTCAGGGATG-3′; RORγt sense, 5′-CCGCTGAGAGGGCTTCAC-3′ and antisense, 5′-TGCAGGAGTAGGCCACATTACA-3′; Foxp3sense, 5′-TGCAGGGCAGCTAGGTACTTGTA-3′ and antisense, 5′-TCTCGGAGATCCCCTTTGTCT-3′; IFN-γ sense, 5′-AGCAACAACATAAGCGTCAT-3′and antisense, 5′-CCTCAAACTTGGCAATACTC-3′; IL-4 sense, 5′-TCCTGCTCTTCTTTCTCG-3′ and antisense, 5′-TTCTCCTGTGACCTCGTT-3′; IL-17sense, 5′-CTCCAGAAGGCCCTCAGACTAC-3′ and antisense, 5′-GGGTCTTCATTGCGGTGG-3′; IL-10 sense, 5′-ACCAAAGCCACAAAGCAG-3′ andantisense, 5′-GGAGTCGGTTAGCAGTATG-3′; β-actin sense, 5′-CTGTCCCTGTATGCCTCTG-3′ and antisense, 5′-ATGTCACGCACGATTTCC-3′.

For semiquantitative PCR, cDNAs were amplified with specificprimers. The oligonucleotide sequences for CCK1 and CCK2 receptorswere as follows: CCK1R sense, 5′-CAGCAGCCGATATGAGGA-3′, and anti-sense 5′-GGCATTGGCACTGAAGAT-3′; CCK2R sense, 5′-ATAGCCTAGTGCGGTAGTGA-3′ and antisense 5′-CTAATGGAAAGGGATGACG-3′. PCRproducts were analyzed using agarose gel electrophoresis withethidium bromide staining. The expected sizes of the amplified frag-mentswere 229 bp for CCK1R, 166 bp for CCK2R, and 218 bp for β-actin.

2.7. Enzyme-linked immunosorbent assay (ELISA)

The concentrations of effector cytokines, IFN-γ, IL-4, IL-17 and IL-10,in cell culture supernatants were analyzed with ELISA, according to themanufacturer's instructions.

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2.8. Statistical analysis

Each experiment was performed in duplicate and repeated at leastthree times. Data are expressed asmean values±SEM. Statistical signif-icance was analyzed with ANOVA, followed by the Student–Newman–Keuls test with SPSS software version 11.5 (SPSS Inc., Chicago, IL). Avalue of P b 0.05 was considered significant.

3. Results

3.1. CCK-8 inhibits Th1 cell differentiation from CD4+ T cells

We initially examined the effects of CCK-8 ondifferentiation of the tra-ditional Th1 and Th2 subsets of CD4+ T cells. CD4+ T cells isolated from

Fig. 1. Effects of CCK-8 on generation of IFN-γ/IL-4-producing cells from CD4+ T cells. CD4+ T cwith plate-boundanti-CD3 and soluble anti-CD28under Th1- or Th2-polarizing conditionswithon fixed cells via intracellular staining. Representative FACS plots of three independent experimand increasing concentrations of CCK-8 (10−10 to 10−6 mol/l) for 4 days. IFN-γ and IL-4 levelswof positive cells ± SEM from three separate experiments. *P b 0.05, compared with medium co

C57BL/6 mice spleens were stimulated under neutral, Th1-polarizing(IL-12, anti–IL-4) or Th2-polarizing (IL-4, anti-IFN-γ) conditions with orwithout CCK-8 (10−10 to 10−6 mol/l) for 2 days in vitro and expandedwith polarizing cytokines and IL-2 for 2 days, again with or withoutCCK-8. IFN-γ and IL-4 expression levels were determined using intracel-lular staining. Our data showed that different polarizing conditions ledto a selective differentiation of Th1 or Th2 cells (Fig. 1A). Moreover,CCK-8 reduced IFN-γ+ T cell production in a dose-dependent mannerbut induced IL-4+ T cells to a slight extent (Fig. 1B).

3.2. CCK-8 inhibits Th17 cells and promotes Treg cell generation

Next, we examined the effects of CCK-8 on Th17 and Treg cell differ-entiation. CD4+ T cells isolated from C57BL/6 mice spleen were

ells were isolated from normal C57BL/6 mice spleenwith MACS. (A) Cells were stimulatedorwithout CCK-8 (10−8 mol/l) for 4 days. IFN-γ and IL-4 expression levelsweremeasuredents are shown. (B) CD4+ T cells were stimulated under Th1- or Th2- polarizing conditionseremeasured on fixed cells via intracellular staining. Data represent themean percentagentrol.

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stimulated under neutral, Th17-polarizing (TGF-β, IL-6, anti-IL-4 andanti-IFN-γ) or Treg-polarizing (TGF-β, anti-IL-4) conditions withor without CCK-8 (10−10 to 10−6 mol/l) for 2 days in vitro, and subse-quently expanded with polarizing cytokines and IL-2 for 2 days with orwithout CCK-8. Expression levels of IL-17 and Foxp3 were analyzed viaintracellular staining. In the absence of CCK-8, polarizing conditions in-duced IL-17+ cells, which were reduced by CCK-8 in a dose-dependentmanner (Fig. 2A andB). Owing to TGF-β1 involvement in the generationof Th17 cells and inducible Treg cells in vitro, it was understandable that

Fig. 2. CCK-8 effects on the generation of IL-17-producing and Foxp3+ cells from CD4+ T ceunder Th17- or Treg-polarizing conditions with or without CCK-8 (10−8 mol/l) for 4 days. IL-1FACSplots of three independent experiments are shown. (B) CD4+ T cellswere stimulated unde10−6 mol/l) for 4 days. IL-17 and Foxp3 levels were measured on fixed cells via intracellular starate experiments. *P b 0.05, compared with medium control.

CCK-8 inhibits IL-17-producing cells, while promoting the generation ofFoxp3-expressing cells (Fig. 2A and B).

3.3. CCK-8 regulates the expression of transcription factors and signaturecytokines

To investigate the mechanism underlying CCK-8 activity on dif-ferentiation of CD4+ T cells, we cultured CD4+ T cell subsets in dis-tinct polarizing conditions in the presence of CCK-8 for 2 days, and

lls. (A) CD4+ T cells were stimulated with plate-bound anti-CD3 and soluble anti-CD287 and Foxp3 levels were measured on fixed cells via intracellular staining. Representativer Th17 or Treg cell- polarizing conditions and increasing concentrations of CCK-8 (10−10 toaining. Data are presented as themean percentage of positive cells ± SEM from three sep-

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analyzed the expression of the specific transcription factors, T-bet,GATA-3, RORγt and Foxp3, and the signature cytokines, IFN-γ, IL-4,IL-17 and IL-10, using real-time RT-PCR. CCK-8 significantlysuppressed transcription of T-bet and RORγt in a dose-dependentmanner, but induced GATA-3 and Foxp3 mRNA expression. Expres-sion of signature cytokines was similar to the results obtainedwith transcription factors. CCK-8 induced a significant decrease inIFN-γ and IL-17 mRNA expression in a dose-dependent manner,

Fig. 3. CCK-8 effects onmRNA expression of transcription factors and signature cytokines in CD4CD28 under Th1, Th2, Th17 or Treg-polarizing conditions and increasing concentrations of CCK-tors (A) and signature cytokines (B) in CD4+ T cell subsetsweremeasuredwith real-time RT-PCmeans ± SEM of three independent experiments. *P b 0.05 and **P b 0.01, compared with me

and conversely, promoted the mRNA levels of IL-4 and IL-10(Fig. 3A and B).

3.4. CCK-8 regulates effector cytokine secretion

CD4+ T cells play an important role in the immune system throughthe generation of distinct cytokine profiles. To evaluate the effects ofCCK-8 on the function of CD4+ T cells, we cultured CD4+ T cell subsets

+ T cell subsets. CD4+ T cells were stimulatedwith plate-bound anti-CD3 and soluble anti-8 (10−10 to 10−6 mol/l). Two days later, the relative expression levels of transcription fac-R using theΔΔCtmethod,withβ-actionmRNAas an internal control. Data are expressed asdium control.

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under distinct polarizing conditions in the presence of CCK-8 for 4 days,and analyzed the secretion of the effector cytokines, IFN-γ, IL-4, IL-17and IL-10, with ELISA. As expected, different polarizing conditions in-duced the selective secretion of effector cytokines. CCK-8 suppressedIFN-γ and IL-17 production by Th1 and Th17 cells, respectively, in adose-dependent manner, but increased IL-10 secretion by Treg cells.However, Th2 cells exerted limited effects on IL-4 secretion (Fig. 4).

3.5. Roles of CCKR in CD4+ T cell differentiation

CCK receptors are pharmacologically classified into two subtypes,CCK1R and CCK2R, according to their affinities for the peptide agonists,CCK and gastrin. We examined CCKR expression in CD4+ T cells usingRT-PCR and subsequent agarose gel electrophoresis. CCK1R and CCK2RmRNA expression was clearly observed in CD4+ T cells (Fig. 5A). Flowcytometry results further demonstrated that CD4+ T cells expressboth CCK1R and CCK2R (Fig. 5B). To evaluate the potential functionalsignificance of CCKR in CD4+ T cell differentiation, we compared the ef-fects of L-364,718 and LY-288,513 (selective antagonists of CCK1R andCCK2R, respectively) on the expression of transcription factors, T-bet,GATA3, RORγt and Foxp3. As expected, both CCK1R and CCK2R antago-nists reduced the suppressive effects of CCK-8 on T-bet and RORγtmRNA, and the stimulatory effects on GATA3 and Foxp3 (Fig. 5C). Ourresults suggest that CCK1R and CCK2R contribute to the effects of CCK-8 on transcription factor expression. Moreover, CCK2R appeared tohave a more significant effect than CCK1R, signifying a role of CCK2Ras a key player in CD4+ T cell differentiation.

4. Discussion

Accumulating evidence has shown that CCK-8 exerts immunomod-ulatory effects onmacrophages, DCs, T cells and B cells, which have pro-tective roles in animal models of KLH-induced atopic pneumonia andcollagen-induced arthritis [27,28]. A previous study showed that theCCK-8 partially exerts its effects by regulating APC function. Here, weexamined the direct effects of CCK-8 on differentiation and effector cy-tokine production of CD4+ T cell subsets in vitro. Notably, CCK-8

Fig. 4. Effects of CCK-8 on cytokine secretion in CD4+ T cell subsets. CD4+ T cells were stimpolarizing conditions and increasing concentrations of CCK-8 (10−10 to 10−6 mol/l). Four daysas means ± SEM of three independent experiments. *P b 0.05 and **P b 0.01, compared with m

suppressed Th1 and Th17 cell differentiation while promoting Tregcell differentiation. However, we observed a limited effect on Th2cells. Similarly, CCK-8 negatively regulated the expression and produc-tion of signature cytokines, IFN-γ and IL-17, by Th1 and Th17 cells, butinduced the expression and secretion of IL-4 and IL-10 by Th2 andTreg cells, respectively. These results were consistent with the effectsof CCK-8 on the expression of the specific transcription factors of dis-tinct CD4+ T cell subsets, which appeared to be mediated mainly byCCK2R.

CD4+ T cells play a critical role in the immune system, particularlythe adaptive immune system. Dysregulated or uncontrolled effector Tcell responses can lead to autoimmunediseases [29]. Since these subsetsperform an increasingly prominent function in many autoimmune andautoinflammatory disorders, regulation of Th1, Th2, Th17 and Treg celldevelopment is of significant interest. Th1 and Th2 subsets are thefirst paradigm for the functional diversification of CD4+ T cells. TheTh1/Th2 imbalance is related to the pathological processes of severaldisorders, such as atopic disease, rheumatic disease, alloimmune re-sponse, infection, and tumors. Rectification of the Th1/Th2 imbalancethus represents an effective therapeutic pathway. In the current study,we demonstrated that CCK-8 suppresses cytokines and master regula-tors for Th1 cells and has a limited positive effect on Th2 cells.The results suggest that CCK-8 may be useful in the regulation of theTh1/Th2 imbalance, particularly towards Th1 dominance. In contrastto the present results, a previous study by our group showed thatCCK-8 stimulates the production of IFN-γ and IL-2, but reduces IL-4and IL-5 generation by KLH-immunized splenocytes [27]. Specifically,CCK-8 enhanced the Th1 response and reduced the Th2 response inthe KLH-immunized mice. Moreover, CCK-8 induced a significant in-crease in the transcription of IFN-γ and T-bet in inguinal draininglymph nodes (DLNs) from the CIA mice, but there were no obviouschanges in GATA-3 [28]. These results suggest that CCK-8 modulatesthe differentiation of CD4+ T cells in vivo by promoting their differenti-ation into Th1 cells. The inconsistent results may be attributable tothe different cell types and culture conditions in the ex vivo studies. Inthe presence of APCs, different cytokines drive naive T cell differentia-tion into different subtypes. IL-12 promotes naive CD4+ T cells to

ulated with plate-bound anti-CD3 and soluble anti-CD28 under Th1, Th2, Th17 or Treg-later, supernatant fractions were collected and measured using ELISA. Data are expressededium control.

Fig. 5. Expression of CCKR in CD4+ T cells and effects on transcription factors of Th1, Th2, Th17 and Treg cells. (A) CD4+ T cells express CCK1R and CCK2RmRNA. Total RNAwas prepared,reverse-transcribed, and amplified in the presence of CCK1R and CCK2R-specific primers and β-actin primers as a loading control. PCR products were analyzed via electrophoresis on a 1%agarose gel containing ethidium bromide. (B) Cytofluorimetric analysis of CCK1R and CCK2R expression in CD4+ T cells. CD4+ T cells were incubated for 30 min at 4 °C with anti-CCK1Rand anti-CCK2R or isotype control Abs. For indirect staining, this step was followed by a second incubation for 30 min at 4 °C with the appropriate anti-isotype FITC-conjugated Abs.(C) Effects of CCK-8 on mRNA expression of transcription factors in CD4+ T cell subsets. CD4+ T cells were stimulated for 2 days under Th1, Th2, Th17 or Treg-polarizing conditions inthe presence or absence of 10−8 mol/l CCK-8, L-364,718 (a CCK1R antagonist) or LY-288,513 (a CCK2R antagonist). Relative expression of T-bet, GATA3, RORγt or Foxp3 mRNA was an-alyzed in CD4+ T cell subsets using real-time RT-PCR. Expression was normalized to β-actin as an internal control. Data are expressed asmeans± SEM of three independent experiments.*P b 0.05 and **P b 0.01, compared with medium control.

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differentiate into Th1 cells by acting on the transcription factors, Stat4and T-bet. CCK-8 promotes IL-12 production in bone marrow-derivedDCs. Therefore, the stimulatory effect of CCK-8 on IFN-γ may beassociated with the modulation of cytokines secreted by APCs. Here,

we have demonstrated that CCK-8 inhibits effector cytokine andtranscription factor production by differentiated Th1, suggesting thatthe peptide exerts direct effects on T cells by suppressing Th1development.

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Th17 and Treg cells are the other two significant CD4+ T cell popula-tions. Th17 cells induced by the cytokines, TGF-β1 and IL-6, secrete theproinflammatory cytokine, IL-17. Overproduction of IL-17 has been ob-served in a variety of models of autoimmune disease, such as experi-mental autoimmune encephalomyelitis and collagen-induced arthritis[30]. Importantly, Th17 cells are involved in the pathogenesis ofhuman allergic and inflammatory diseases, including asthma, multiplesclerosis, rheumatoid arthritis and inflammatory bowel disease [31].iTreg (CD4+CD25+Foxp3+) cells suppress the responses of APC andeffector T cells through direct interactions or secretion of anti-inflammatory cytokines, such as IL-10 and TGF-β in the periphery, inwhich Treg cells play a critical role in resisting inflammation and regu-lating adaptive immunity to a variety of pathogens [32]. Therefore, thepotential ability to influence Th17/Treg development has attracted sig-nificant clinical interest [31,33,34]. Alteration of the Th17/Treg ratio infavor of increased numbers of Treg cells would be beneficial for a widerange of autoimmune and autoinflammatory diseases, while the inhibi-tion of Treg cell formation is a potential strategy for tumor immunother-apy [30]. In this study, we compared the effects of CCK-8 on thedifferentiation of Tregs mediated by TGF-β and the generation of Th17cells induced by TGFβ and IL-6 in vitro. CCK-8 promoted the differenti-ation of Tregs while potently inhibiting Th17 cell generation. The recip-rocal activity of CCK-8 in inhibiting the production of pro-inflammatoryTh17 cells and promoting anti-inflammatory Treg cell development in-dicates that CCK-8 modulates the balance between pro- and anti-inflammatory immunity. Our data support the utility of CCK-8 as anovel agent for the treatment of inflammatory and autoimmunediseases.

Transcription factors are essential for CD4+ T cell differentiation andcytokine production. Activation, development and expansion of CD4+ Tcells are tightly modulated by the activity and expression of specifictranscription factors [35]. T-bet is a transcription factor responsible forTh1 cell development. An earlier study showed that T cells lose the abil-ity to produce IFN-γ in T-bet-deficientmice, while retroviral expressionof T-bet in developing and developed Th2 cells not only induces IFN-γproduction but also inhibits IL-4 and IL-5 generation [36]. GATA-3, aTh2 cell transcription factor, is critical for the transactivation of Th2 cy-tokine genes aswell as the inhibition of Th1development through an IL-4-independent mechanism [37,38]. The transcription factor RORγt hasbeen shown to be involved in gene transcription of Th17 cells [9]. Over-expression of RORγt promotes IL-17 generation, whereas its deficiencyvirtually abrogates Th17 development [39]. The transcription factorFoxp3 regulates thedifferentiation and function of natural and inducibleCD4+CD25+Treg. To clarify the effects of CCK-8 on CD4+ T cell differen-tiation,we examined T-bet, GATA3, RORγt and Foxp3mRNA expressionunder Th1, Th2, Th17 or Treg cell culture conditions with or withoutCCK-8. The effects of CCK-8 on Th1, Th2, Th17 and Treg cell differentia-tion correlated with the observed inhibition of T-bet and RORγt expres-sion and enhancement of GATA-3 and Foxp3 expression.

CCK-8 exerts pleiotropic activity via binding to CCK1R and CCK2R. Tofurther elucidate the receptor family critically implicated in the directeffects of CCK-8 on functional differentiation of CD4+ T cells, we exam-ined CCK1R and CCK2RmRNA and protein expression inMACS-purifiedCD4+ T cells, and used selective antagonists of CCK1R and CCK2R toevaluate the expression of transcription factors, T-bet, GATA, RORγtand Foxp3. Our results showed that CCKR antagonists partially blockthe inhibition of T-bet and RORγt expression and enhancement ofGATA3 and Foxp3 expression induced by CCK-8, supporting a role ofCCK-8 in regulating the balance between Th1 and Th2 or Treg andTh17 differentiation. Based on the CCKR expression data and the effectsof selective antagonists, we propose that CCK-8 acts via CCK1R andCCK2R to directly affect CD4+ T cell development. Furthermore,CCK2R is possibly the main candidate mediating this effect.

In summary, CCK-8 inhibits Th1 and Th17 polarization while pro-moting Treg differentiation. Our data show that CCK-8 plays an impor-tant role in the regulation of T-cell lineage development. Moreover,

CCK-8 and CCK receptor antagonists are potentially useful pharmaco-logic agents for themanagement of inflammatory and immunediseases.These insightsmay lead to the development of novel approaches for thetreatment of inflammatory diseases and immune disorders. However,further research is required to elucidate the precise roles of CCK-8 innormal CD4+ T cell differentiation and function.

Acknowledgments

This work was supported by grants from the China PostdoctoralScience Foundation funded project (No. 20080440822).

Declaration of interest: The authors report no conflict of interest.The authors alone are responsible for the content and writing of thepaper.

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