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Molecular Immunology 51 (2012) 150– 158

Contents lists available at SciVerse ScienceDirect

Molecular Immunology

j ourna l ho me pag e: www.elsev ier .com/ locate /mol imm

haeoglobosin Fex inhibits poly(I:C)-induced activation of bone marrow-derivedendritic cells

in Suna, Chunyan Huaa, Yonghong Yanga, Huan Doua, Erguang Lia, Renxiang Tanb,∗, Yayi Houa,∗

Immunology and Reproductive Biology Lab & Jiangsu Key Laboratory of Molecular Medicine, School of Medicine, Nanjing University, Nanjing 210093, PR ChinaInstitute of Functional Biomolecules, State Key Laboratory of Pharmaceutical Biotechnology, School of Lifesciences, Nanjing University, Nanjing 210093, PR China

r t i c l e i n f o

rticle history:eceived 14 December 2011eceived in revised form 22 February 2012ccepted 28 February 2012vailable online 17 March 2012

eywords:haeoglobosin Fex

a b s t r a c t

Dendritic cells (DCs) are implicated in the induction of autoimmune diseases and exist in lesions associ-ated with several autoimmune inflammatory diseases. Chaeoglobosin Fex (Cha Fex), a cytochalasan-basedalkaloid, was isolated from marine-derived endophytic fungus Chaetomium globosum QEN-14. In thepresent study, we evaluated the effect of Cha Fex on poly(I:C)-induced bone marrow-derived DCs. Theresults showed that Cha Fex attenuated the production of IFN-� both at the mRNA and protein level inpoly(I:C)-induced DCs. Cha Fex markedly inhibited the maturation and function of the DCs with a reducedcapacity to uptake antigens and low level of expression of costimulatory molecules. Moreover, Cha Fex

MDCsaturation

LR3 signalingFN-�

abrogated the ability of poly(I:C)-induced DCs to promotion of T cell proliferation, Furthermore, Cha Fexinhibited the phosphorylation of I�B-� and IRF-3 in poly(I:C)-induced DCs. Cha Fex also reduced thephosphorylation of p38 and JNK, without affecting ERK1/2. These data demonstrate that that Cha Fex canexhibit an immunosuppressive effect on mouse bone marrow-derived DCs (BMDCs) via TLR3 signaling,which suggests potential application of Cha Fex in the treatment of autoimmune inflammatory diseases.

. Introduction

DCs are the most potent professional antigen presenting cellsAPCs) and play a crucial role at the cross-talk of innate and adaptivemmune systems (Merad and Manz, 2009). Substantial evidencendicates that DCs are implicated in the induction of autoim-

une diseases and positioned in lesions associated with severalutoimmune inflammatory diseases (Bennett and Chakraverty,011; Capini et al., 2009; Du et al., 2011; O’Sullivan et al., 2011).mportantly, DCs become mature, the expressions of costimula-ory molecules such as MHC, CD40, CD80 or CD86 on their surfacesre upregulated, and a variety of chemokines and cytokines areroduced in response to a spectrum of stimuli through activationf their TLRs (Akira et al., 2006; Reis e Sousa, 2004). Among the

0 known human TLRs, TLR3 is responsible for sensing dsRNA-aommon byproduct or intermediate in viral genome replicationAlexopoulou et al., 2001). TLR3 signaling is TRIF-dependent in

Abbreviations: DCs, dendritic cells; Cha Fex, Chaeoglobosin Fex; BMDCs, bonearrow-derived dendritic cells; APCs, antigen presenting cells; IL-6, interleukin-6;

L-12, interleukin-12; TNF-�, tumor necrosis factor-�; MCP-1, monocyte chemoat-ractant protein-1; Cha K, Chaetoglobosin K; PI, propidium iodide; MLR, mixedymphocyte reaction; IDDM, insulin-dependent diabetes mellitus.∗ Corresponding authors. Tel.: +86 25 83686441; fax: +86 25 83686441.

E-mail addresses: yayihou@nju.edu.cn (R. Tan), rxtan@nju.edu.cn (Y. Hou).

161-5890/$ – see front matter © 2012 Elsevier Ltd. All rights reserved.oi:10.1016/j.molimm.2012.02.125

© 2012 Elsevier Ltd. All rights reserved.

contrast to other TLR signalings except TLR4 signaling (Lee and Kim,2007; Matsumoto et al., 2011), namely TLR3 signaling and is initi-ated by the recruitment of TRIF, which interacts with TRAF3, TBK1and IKKi. TBK1, together with IKKi, mediates phosphorylation ofIRF3, which then dimerizes and translocates to the nucleus whereit activates type I interferon promoters, especially IFN-� promoter.

DCs are capable of producing IFN-� in response to viral infec-tion and viral dsRNA mimicking polyinosinic:polycytidylic acid(polyI:C). On the other hand, IFN-� is known to have the effectof DC function and maturation. IFN-� can induce the productionof IFN-inducible genes (Theofilopoulos et al., 2005). Besides IRF3signaling, TRIF-RIP1 and TRIF-TRAF6 signaling in the TLR3 pathwayinduce NF-�B and MAPK activation, resulting in NF-�B translocateto the nuclei and phosphorylation and activation of AP-1 respec-tively, both of which contribute to the transcriptional induction ofproinflammatory cytokines and chemokines, such as interleukin-6 (IL-6), interleukin-12 (IL-12), tumor necrosis factor-� (TNF-�),monocyte chemoattractant protein-1 (MCP-1). Moreover, previousstudies showed that TLR3 pathway also takes part in many diseaseprogressions, so it is a promising therapeutic target for the treat-ment of these diseases (Gauzzi et al., 2010; Gowen et al., 2006;Hutchens et al., 2008; Li et al., 2009).

The chaetoglobosins are a series of cytochalasins with novelstructures and biological activities. To date, some chaetoglobosinshave shown a wide range of biological functions. Chaetoglo-bosin K (Cha K), for example, has inhibitory effects on Akt kinase

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hosphorylation and cytokinesis in ras-transformed cells (Matesict al., 2006). Cha K also has the preventative and reversal effectsn lindane and dieldrin inhibition of gap junction-mediated com-unication (Sidorova and Matesic, 2008). Chaeoglobosin Fex (Cha

ex) is a cytochalasin isolated from Chaetomium globosum QEN-4. QEN-14 is a marine green alga UlVa pertusa. Our recent studyhowed that Cha Fex has inhibitory effects on LPS-induced TNF-, IL-6 and MCP-1 production through blocking the degradationf I�B-� and the phosphorylation of JNK, ERK1/2, and p38. Theseesults suggest that Cha Fex has the potential to repress TLR4 sig-aling in macrophages activated directly by LPS stimulation (Dout al., 2011). However, it is still unclear whether Cha Fex possesseshe effect on the professional antigen presenting cells for antigenetection and its stimulation function to other effector lympho-ytes.

The aim of this study was focused on the immunoregula-ory properties of Cha Fex on poly(I:C)-induced activation ofLR3-mediated signaling in DCs. Our present study demon-trated that Cha Fex significantly decreased the production ofro-inflammatory cytokines and the expression of costimulatoryolecules in poly(I:C)-induced DCs. Cha Fex also attenuated the

apability of mature DCs to drive T cell proliferation. Moreover,ha Fex down-regulated the IFN-� production and inhibited thectivation of IRF-3, NF-�B, p38 and JNK signaling pathways. Thesendings confirmed that Cha Fex can acts as a negative regulatorf poly(I:C)-induced activation of DCs. This suggests that Cha Fexay be a promising compound for the treatment of TLR3-mediated

utoimmune and inflammatory diseases.

. Materials and methods

.1. Reagents

Synthesis and chemical structure of Chaeoglobosin Fex werereviously reported (Dou et al., 2011). RPMI 1640, fetal bovineerum (FBS), penicillin and streptomycin were purchased fromibco Inc (Grand Island, NY, USA). Recombinant GM-CSF wasurchased from Miltenyi Biotech (Bergisch Gladbach, Germany).ecombinant IL-4 was purchased from Peprotech, Inc. (Rocky Hill,J). Poly(I:C) was obtained from Invitrogen (San Diego, CA, USA).ITC Annexin V Apoptosis Detection Kit was purchased from BDharmingen (Heidelberg, Germany). Mouse Interferon Beta ELISAit was purchased from PBL Biomedical Laboratories (Piscataway,J, USA). APC-conjugated anti-CD40, APC-conjugated anti-MHCII,E-conjugated anti-CD11c, PE-conjugated anti-CD80, and PE-onjugated anti-CD86 from eBioscience (San Diego, CA, USA). BCAit was purchased from Pierce (Rockford, IL). FITC-dextran fromigma–Aldrich (St Louis, MO, USA). Antibodies for anti-mouse JNK,hospho-JNK, p38, phospho-p38, ERK1/2, phospho-ERK1/2, I�B-�,hospho-I�B-�, IRF-3, phospho-IRF-3 and GAPDH for western blotere ordered from Cell Signaling Technology (Danvers, MA, USA).

.2. Mice

Female C57BL/6 (B6) and Balb/c mice 4–6 weeks old were pur-hased from the Animal Research Center of Yangzhou University, PRhina. The animals were acclimatized for 1 week before the studynd fed on standard rodent chow and water. The mice were main-ained under a 12-h light/12-h dark cycle, at 20–22 ◦C and with0–60% humidity levels.

.3. Generation of bone marrow derived dendritic cells

DCs were generated as previously described (Xie et al., 2011). Inrief, bone marrow was isolated from the tibiae and femora of mice,

logy 51 (2012) 150– 158 151

processed with red blood cell lysing buffer and cultured in RPMI-1640 medium supplemented with 10% FBS, 100 �g/ml penicillin,100 �g/ml streptomycin, 10 ng/ml murine GM-CSF and 1 ng/ml IL-4 in a humidified atmosphere of 5% CO2 at 37 ◦C for 7 days. Half ofthe medium was replaced by fresh medium containing GM-CSF andIL-4 on days 3 and 5, then the loosely adherent cells were dislodgedand harvested gently, and more than 85% of the cells were CD11cpositive. Bone marrow derived DCs were cultured in new cultureplates for further experiments.

2.4. Cell viability

The effect of Cha Fex on cell viability of DCs was evaluated by theAnnexin V/PI staining. Briefly, mouse DCs were cultured for 24 h or48 h with various concentrations of Cha Fex at 37 ◦C. Then cells werewashed in cold PBS, incubated with Annexin V in binding buffer. PIwas added prior to analysis with flow cytometry. Flow cytometrywas performed by using FACSCalibur (Becton Dickinson, San Jose,CA, USA) and the data were analyzed by WinMDI (Scripps Institute,La Jolla, CA).

2.5. IFN- ̌ ELISA

DCs were treated with poly(I:C) in the absence or presenceof various concentrations of Cha Fex. Supernatants were har-vested after 24 h and assayed for IFN-� using the enzyme-linkedimmunosorbent assay kit according to the instructions of the man-ufacturers. Briefly, cell culture supernatants and IFN-� standardsin triplicate were added to pre-coated microtiter plate and incu-bated for 60 min in a closed chamber at 22–25 ◦C. Afterward, theplate was washed three times with prepared wash solution. Thenantibody was added and the plate was incubated for 60 min at22–25 ◦C. After washing again, HRP solution was added and theplate was incubated for 60 min at 22–25 ◦C. The plate was washedthree times again, TMB substrate solution added and incubated for15 min in the dark. At last, stop solution was added to each welland the absorbance was determined at 450 nm within 5 min usinga microplate reader (BioTek).

2.6. RNA extraction and real-time quantitative polymerase chainreaction analysis

Total RNA was extracted from 2 × 106 cells using TRIZOL (Invit-rogen, Carlsbad, CA, USA). RNA isolation and cDNA synthesis wereperformed as described previously (Xie et al., 2011). The mRNAlevels for target gene were quantified by real-time quantitativepolymerase chain reaction (qPCR) by using the 7300 Real-TimePCR system (Applied Biosystems, Foster City, Calif., USA). PCR wassetup by mixing 1 �l of cDNA template, 1 �l of 500 nM forward andreverse primer and 10 �l of FastStart Universal SYBR Green Master(Rox) (Roche, Mannheim, Germany) in 20 �l PCR reaction mixture.Amplification conditions were 55 ◦C for 2 min, 95 ◦C for 10 min, and40 cycles of 95 ◦C for 30 s, and 60 ◦C for 30 s. The expressions ofgenes (fold change versus medium control) were normalized using�-actin as the internal control. Each sample was run in triplicate andthe experiment was repeated three times or more. Mouse primersequences used in this experiment are indicated in Table 1 andsynthesized by Invitrogen.

2.7. Endocytosis

Endocytotic activity of DCs was measured as cellular uptake of

FITC-dextran and was quantified by flow cytometry. Briefly, DCswere treated with various concentrations of Cha Fex for 2 h, washedand incubated with 0.5 mg/ml FITC-dextran in the dark for 1 h at37 ◦C. DCs incubation with FITC-dextran for 1 h at 4 ◦C served as

152 L. Sun et al. / Molecular Immunology 51 (2012) 150– 158

Table 1Primers used for qPCR studies in BMDCs.

Primer Sequence

IFN-� F 5′-CAGCTCCAAGAAAGGACGAAC-3′

IFN-� R 5′-GGCAGTGTAACTCTTCTGCAT-3′

IP-10 F 5′-CCAAGTGCTGCCGTCATTTTC-3′

IP-10 R 5′-GGCTCGCAGGGATGATTTCAA-3′

IL-6 F 5′-TGCCTTCTTGGGACTGAT-3′

IL-6 R 5′-TAAGCCTCCGACTTGTGA-3′

IL-12 F 5′-AGACATGGAGTCATAGGCTCTG-3′

IL-12 R 5′-CCATTTTCCTTCTTGTGGAGCA-3′

TNF-� F 5′-CCCTCACACTCAGATCATCTTCT-3′′ ′

aceb

2

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2

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2

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2

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Fig. 1. Effect of Cha Fex on the cell viability of BMDCs. Bone marrow-derived den-dritic cells from C57BL/6 mice were incubated with indicated concentrations of Cha

TNF-� R 5 -GCTACGACGTGGGCTACAG-3MCP-1 F 5′-TTAAAAACCTGGATCGGAACCAA-3′

MCP-1 R 5′-GCATTAGCTTCAGATTTACGGGT-3′

negative control. Then, the cells were washed three times withold PBS, resuspended with 1.2 mg/ml trypan blue to quench thendocytic activity and remove any free FITC-dextran, and analyzedy flow cytometry.

.8. Analysis of the effect of Cha Fex on uptake of poly(I:C)

The uptake of poly(I:C) by DCs was monitored by flow cytom-try using poly(I:C) conjugated to rhodamine. In brief, cells werere-treated with or without Cha Fex for 2 h before incubatedith poly(I:C)-rhodamine for 24 h. Then washed twice with PBS

o remove any free poly(I:C)-rhodamine and analyzed by flowytometry. All the results were expressed as median fluorescencentensity (MFI).

.9. Flow cytometry analysis of surface markers

DCs were stained with the fluorescently labeled monoclonalntibodies or isotype control antibodies at 4 ◦C for 30 min in theark. Fluorochrome-conjugated anti-mouse antibodies with theollowing specificities were used: IgG1 and IgG2a isotype controls,nti-CD40, CD80, CD86, and MHCII. The cells were then washedith PBS and fixed in PBS containing 1% paraformaldehyde. Cellsere detected by flow cytometry. All the results were expressed asedian fluorescence intensity (MFI).

.10. Mixed lymphocyte reaction

Stimulatory capacity of DCs to T cells was reflected in the allo-enic MLR assay. Allogeneic T cells (5 × 105/well) as responder cellsrom BALB/c mice were cocultured in triplicate with DCs (DC:T cellatios of 1:10, 1:40 and 1:100). MLR was conducted in a 96-wellat-bottom microplate in 200 �l complete RPMI 1640 with 10%FBS

n 5%CO2 incubator at 37 ◦C for 3 days. Then the proliferation of Tells was monitored by 3H incorporation assay.

.11. Western immunoblot analysis

DCs were pretreated with or without Cha Fex, then stimulatedith poly(I:C) at different time points. After that, the cells wereashed with twice with ice-cold phosphate buffered saline (PBS),

nd lysed as described previously (Xie et al., 2011). Protein concen-rations were measured using the BCA Protein Assay Reagent Kit.qual amounts of protein from each sample were separated by a0% SDS-polyacrylamide gel electrophoresis and transferred ontoolyvinylidene difluoride membranes (Roche, Germany). After theVDF membranes were blocked for 1 h in TBST (25 mM Tris–HCl,

H 7.6, 125 mM NaCl, 0. 1% Tween-20) containing 5% bovineerum albumin (BSA), the membranes were incubated overnightith primary antibodies against rabbit anti-mouse phospho-p38,hospho-ERK1/2, phospho-IRF3, phospho-I�B-�, p38, ERK, JNK,

Fex for 24 h or 48 h. The viability of murine BMDCs was assessed with Annexin V/-FITC apoptosis detection kit by flow cytometry. Annexin V−/PI− cells were viableand not undergoing apoptosis. *P < 0.05 vs. untreated group.

IRF3, I�B-�, GAPDH mAb and mouse anti-phospho-JNK antibody at1:1000 dilutions in antibody dilution buffer (5% BSA in TBST) withgentle shaking at 4 ◦C. Subsequently, the membranes were incu-bated HRP conjugated goat anti-rabbit or anti-mouse antibodies assecond antibodies at 1:3000 dilutions for 2 h at the room tempera-ture. The membranes were washed with TBST again. Protein bandswere developed by enhanced chemiluminescence Western blottingdetection reagents, and the images were taken by FluorChem FC2System (Alpha Innotech, USA). The membrane was stripped andreprobed with GAPDH antibody to verify equal loading.

2.12. Statistical analysis

All results were expressed as mean ± SD from three or moreindependent experiments and analyzed with Prism 4 (GraphPadSoftware, Inc., San Diego, CA). Student’s t-test and one-way ANOVAanalysis of variance were used to compare between different treat-ments. Differences were considered statistically significant whenthe value of P was <0.05.

3. Results

3.1. Cha Fex inhibited expression of IFN- ̌ in poly(I:C)-inducedDCs

BMDCs did not proliferate in vitro, so we first examined theeffect of Cha Fex on the cell viability of DCs by Annexin V/PI staining.No significant difference was detected in the cells treated with orwithout Cha Fex in the range of 0–4 �M for 24 h or 48 h (Fig. 1).When the concentration was raised to 8 �M, there was a sharpfall in cell viability both at 24 h and 48 h. Annexin V/PI stainingof BMDCs indicated that Cha Fex had no cytotoxicity to DCs at theconcentrations low to 8 �M.

DCs can produce IFN-� in response to viral infection and viraldsRNA mimicking poly(I:C). IFN-� can also increase expression ofcostimulatory molecules leading to DC maturation (Theofilopouloset al., 2005). Thus the change of IFN-� expression may feature thematuration and function of DCs. Here, the expression of IFN-� wasanalyzed to clarify the effect of Cha Fex on poly(I:C)-induced acti-vation of DCs. DCs were cultured either in the presence or in theabsence of Cha Fex, the cell-free culture supernatant was collected24 h after poly(I:C) stimulation, and the amount of IFN-� was ana-lyzed by ELISA. As shown in Fig. 2, the cells treated with Cha Fex and

poly(I:C) produced lower amounts of IFN-�, compared with thosewith poly(I:C) alone. Moreover, Cha Fex impaired IFN-� productionof poly(I:C)-induced DCs in a does-dependent manner. The similarresult was found in the transcription level of IFN-� (Fig. 2B).

L. Sun et al. / Molecular Immunology 51 (2012) 150– 158 153

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ig. 2. Effect of Cha Fex on poly(I:C)-induced expression of IFN-� on BMDCs. BMDCfor real-time PCR) or 24 h (for ELISA). The culture supernatants were collected andFN-� with real-time PCR. The data are shown as the mean ± SD. #P < 0.05 vs. untrea

.2. Cha Fex attenuated the phenotypic maturation ofoly(I:C)-induced DCs

The maturation process of DCs is characterized by the inductionf MHCII and high expression of costimulatory molecules such asD40, CD80 and CD86. Profile of cytokines expression and pheno-ypic maturation of DCs are differentially depended on stimulationf TLR ligands (Hoebe et al., 2003; Jones et al., 2010; Warger et al.,006). To investigate if Cha Fex modulated the maturation of DCs,he expressions of MHC II and costimulatory molecules on DCsere examined by Flow cytometry. After exposure to poly(I:C)

25 �g/ml) for 24 h, the expressions of MHCII, CD40, CD80 andD86 on DCs were upregulated, while the expressions were signif-

cantly inhibited with pretreatment of Cha Fex (Fig. 3A–D). Cha Fexlone did not influence the expression of these molecules comparedith that in control group. The suppression of CD86 expression

n poly(I:C)-induced DCs was observed at 0. 5 �M Cha Fex, whilenhibitory effect for CD80 reached at higher concentration at 2 �Mha Fex. These results indicated that Cha Fex suppressed the mat-ration of poly(I:C)-induced DCs, which suggests the ability of theCs to present antigen to T cells was impaired.

.3. Cha Fex reduced the expression of genes encodingnflammatory cytokines and chemokines in poly(I:C)-induced DCs

Excepting IRF3-mediated IFN-� induction, the activation of TLR3athway also controlled the expression of genes encoding inflam-atory cytokines and chemokines such as IP-10, IL-6, IL-12, TNF-�

nd MCP-1 through the activation of some critical transcriptionactors – NF-�B and AP-1 (Gauzzi et al., 2010). IP-10 is one of theFN-induced proteins and plays an important role in the interactionetween DCs and T cells through binding and activating the sevenransmembrane G protein coupled receptor CXCR3 expressed in Tells (Qian et al., 2007). We found that Cha Fex markedly impairedhe expression of IP-10 mRNA in DCs following poly(I:C) stimula-ion (Fig. 4). Moreover, Cha Fex also decreased the levels of IL-6,L-12, TNF-� and MCP-1 mRNA in poly(I:C)-induced DCs comparedo those in control (Fig. 4). These data suggested that Cha Fex ham-ered the signal transduction of TLR3 pathway in DCs.

.4. Cha Fex interfered the endocytic function ofoly(I:C)-induced DCs

Endocytic capacity reflects the efficient eradication of immatureCs against pathogen (Schakel, 2009). FITC-dextran is generallysed to examine the intrinsic phagocytosis capacity. As shown

n Fig. 5A, DCs pretreated with Cha Fex for 2 h exhibited a lowerndocytic capacity than that in normal DCs at 37 ◦C. The endocyticctivity of DCs showed a negative correlation with the concentra-ion of Cha Fex. Furthermore, TLR3 is expressed intracellularly and

e pretreated with Cha Fex for 2 h, prior to poly(I:C) stimulation at 25 �g/ml for 6 hyed for IFN-� with ELISA, or the RNA was extracted to measure the mRNA level ofoup and *P < 0.05 vs. responses in the absence of Cha Fex with poly(I:C) stimulation.

localized to the endosomal compartment of myeloid DCs. To clar-ify whether Cha Fex affects uptake of DCs for poly(I:C), poly(I:C)conjugated to rhodamine (poly(I:C)-rhodamine) was utilized todetermine the distribution of poly(I:C) in DCs. The results showedthat DCs pretreated with Cha Fex did not exhibit a remarkablereduction in endocytic capacity in exposure to poly(I:C)-rhodamineat 37 ◦C for 24 h (Fig. 5B). These data demonstrated that ChaFex interferes the endocytic function of DCs and implied thatthe decreased expression of IFN-�, inflammatory cytokines andchemokines may be not related to uptake of poly(I:C) in DCs.

3.5. Cha Fex suppressed the stimulatory capacity of TLR3-inducedDCs to T cells

The immunostimulatory properties of DCs to T cells are essentialfor adaptive immune responses. Here allogenic mixed lympho-cyte reaction (MLR) was performed to determine whether Cha Fexmodulate the ability of poly(I:C)-induced DCs to induce the pro-liferation of allogenic T cells. Cha Fex alone at a concentration of2 �M did not significantly affect the ability of DCs induce the pro-liferation of T cells compared to non-treated counterparts, whileCha Fex inhibited the ability of poly(I:C)-induced DCs to stimulateallogenic T cells at all ratios of T cell:DC tested (Fig. 6). These datasuggest that Cha Fex may reduce the immunostimulatory capacityof poly(I:C)-induced DCs to effector lymphocytes, which play keyrole in adaptive immune responses or inflammatory diseases.

3.6. Cha Fex pretreatment suppressed TLR3-mediated signalingpathways in DCs

The activation of IRF-3, NF-�B and MAPKs is important forimmune system responses in the TLR3-dependent signaling path-ways (Kawai and Akira, 2006). In order to further explore theunderlying mechanism for the inhibitory role of Cha Fex on DCs,the activation of IRF-3, NF-�B and MAPKs was examined. DCs werepretreated with Cha Fex at 2 �M for 2 h followed by poly(I:C)stimulation at 25 �g/ml at different time points and whole celllysates were analyzed by immunoblotting. As shown in Fig. 7Aand B, Cha Fex potently inhibited the phosphorylation of IRF-3and I�B induced by poly(I:C), while poly(I:C)-induced I�B degra-dation was abolished by Cha Fex. Similar results were found forMAPK pathways. In addition, Cha Fex pretreatment also decreasedphosphorylation of the kinases JNK and p38 induced by poly(I:C),whereas their total protein levels were unaffected (Fig. 7C).

4. Discussion

In the study, we found that Cha Fex attenuated the production ofIFN-� both at the mRNA and protein level in poly(I:C)-induced DCs.Cha Fex markedly inhibited the maturation and function of the DCs

154 L. Sun et al. / Molecular Immunology 51 (2012) 150– 158

Fig. 3. Cha Fex inhibits poly(I:C)-induced upregulation of surface molecules on BMDCs. After pretreated with the indicated concentrations of Cha Fex for 2 h, BMDCs werestimulated with 25 �g/ml poly(I:C) for 24 h. The expressions of MHCII (A), CD40 (B), CD80 (C) and CD86 (D) were analyzed by flow cytometry. The mean fluorescence intensity(MFI) values are shown as the mean ± SD. #P < 0.05 vs. untreated group and *P < 0.05 vs. responses in the absence of Cha Fex with poly(I:C) stimulation.

L. Sun et al. / Molecular Immunology 51 (2012) 150– 158 155

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ig. 4. Effect of Cha Fex on poly(I:C)-induced expression of genes encoding inflam h, prior to poly(I:C) stimulation at 25 �g/ml for 6 h. The RNA was extracted to meare shown as the mean ± SD. #P < 0.05 vs. untreated group and *P < 0.05 vs. response

ith a reduced capacity to uptake antigens and low level of expres-ion of costimulatory molecules. Moreover, Cha Fex abrogated thebility of poly(I:C)-induced DCs to promotion of T cell proliferation,urthermore, Cha Fex inhibited the phosphorylation of I�B-� andRF-3 in poly(I:C)-induced DCs. Cha Fex also reduced the phospho-ylation of p38 and JNK, without affecting ERK1/2. Cha Fex maybe

potential drug in the treatment of TLR3-mediated diseases.DCs are potent antigen presenting cells and play a funda-

ental role in linking innate and adaptive immunity. Moreover,edical implications of DCs biology have been extended to the

iagnosis, management and prevention of diseases (Steinman andanchereau, 2007). The recognition of TLR3 to poly(I:C) elicits the

nductive expression of type I interferons, which plays a dominant

ig. 5. Effect of Cha Fex on endocytic function of BMDCs. (A) Control and Cha Fex treated

white bars) followed by an endocytosis assay. (B) To investigate whether Cha Fex effect Boly(I:C)-rhodamine was then added to the culture and incubated for an additional 24 h. The mean ± SD. *P < 0.05 vs. untreated group.

y cytokines and chemokines on BMDCs. BMDCs were pretreated with Cha Fex forhe mRNA level of IP-10, IL-6, IL-12, TNF-� and MCP-1 with real-time PCR. The datahe absence of Cha Fex with poly(I:C) stimulation.

role in shaping downstream events of DCs such as maturation,expression of surface molecules, and production of inflammatorycytokines and chemokines. In this study, we found that Cha Fexpotently decreased the expression level of IFN-� mRNA and proteinin poly(I:C)-induced DCs. Many previous studies showed that IFNstake part in several autoimmune diseases, like lupus and insulin-dependent diabetes mellitus (IDDM) (Nguyen-Pham et al., 2011;Theofilopoulos et al., 2005). This suggests that Cha Fex may beinstrumental in immunoregulation.

Cha Fex markedly inhibited the maturation and function of the

DCs. There are two major states during the development of DCs. Thematuration state of DCs is a critical determinant of the developmentof their immune responses. While immature DCs, characterized

BMDCs were incubated with FITC-dextran for 2 h at either 37 ◦C (black bars) or 4 ◦CMDCs to take up poly(I:C) or not, BMDCs were pre-treated with Cha Fex for 2 h, andhe cells were collected and analyzed with flow cytometry. MFI value are shown as

156 L. Sun et al. / Molecular Immunology 51 (2012) 150– 158

Fig. 6. Cha Fex inhibits the capability of stimulating T-cell response in MLR of poly(I:C)-induced BMDCs. In MLR experiments, immature BMDCs or mature BMDCs werep T cele ean ±F

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retreated with Cha Fex and used in graded cell numbers to stimulate allogeneicvaluated by incubating cells with 3H-thymidine assay. The data are shown as the mex with poly(I:C) stimulation.

y expression of low surface levels of MHC II and costimulatoryolecules, are avidity endocytic and poor in inducing T cell prim-

ng. DCs activated by TLRs ligands, like poly(I:C), are monitoredy elevated expression of costimulatory molecules and high-level

ig. 7. Cha Fex inhibits the activation of TLR3-mediated signaling pathway. BMDCs were phe cells were extracted to determine the effect of Cha Fex priming on poly(I:C)-induced are representative of three independent experiments or more.

ls (5 × 105 responder cells/well). After culture for 3 days, T-cell proliferation was SD. #P < 0.05 vs. untreated group and *P < 0.05 vs. responses in the absence of Cha

expression of proinflammatory cytokines and chemokines, andinduce T cells activation and effectors differentiation (Boes et al.,2004; Wilson et al., 2006; Winzler et al., 1997; Kato et al., 2000;Lipscomb and Masten, 2002). Our present data showed that Cha Fex

re-exposed to Cha Fex at 2 �M for 2 h, followed by poly(I:C) treatment at 25 �g/ml.ctivation of IRF-3 (A), NF-�B (B), MAPK (C) pathways by immunoblot analysis. Data

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L. Sun et al. / Molecular Im

own-regulated the expression of CD40, CD80, CD86 and MHC II inoly(I:C)-induced DCs. Cha Fex strongly down-regulated expres-ion of IL-6 IL-12, IP-10, TNF-� and MCP-1 on transcription level.n the other hand, the capability of uptake of immature DCs wasttenuated, suggesting that the DCs could not utilize membraneeceptors, like MMP to take up FITC-dextran, a carbohydrate-onjugated antigen effectively. In our study, DCs treated with Chaex for 2 h showed similar uptake ability of poly(I:C)-rhodamineo that in control. As known, poly(I:C) is internalized via clathrin-ependent endocytosis to activate TLR3 in endosome (Itoh et al.,008). Therefore, these results further indicated that Cha Fexeduced TLR3-mediated DCs activation through inhibiting surfaceolecules expression, cytokines production and partially blocking

he maturation of DCs, but not the uptake of poly(I:C).Cha Fex abrogated the ability of poly(I:C)-induced DCs to promo-

ion of T cell proliferation, DCs activate naïve T cells due to their highurface expression of MHC and costimulatory molecules (Proiettot al., 2008). To further confirm the inhibitory effect of Cha Fex onCs function, we performed MLR to assess whether mature DCs

nduced by poly(I:C) can activate allogenic T cells to proliferateffectively. The results indicated that Cha Fex partially inhibitedCs maturation, resulting in attenuating DCs ability of activatingroliferation of T cells. These results revealed that Cha Fex may be

potent inhibitory mediator of DC maturation.Cha Fex can exhibit an immunosuppressive effect on mouse

one marrow-derived DCs via TLR3 signaling. Given the fact thatha Fex could inhibit the phenotypic and functional matura-ion of DCs, it was necessary to explore the mechanism involvedn anti-inflammatory properties of Cha Fex. As is known, TLR3-

ediated immune response is a MyD88-independent cellularmmune response, which recruits TRIF in response to poly(I:C) torigger downstream signals through activating NF-�B, IRF-3 and

APKs and initiate transcription of inflammatory cytokines. IRF- is essential for IFN-� induction, while with the phosphorylated�B-� ubiquitinated and degraded, NF-�B translocates into theucleus and initiates the transcription of inflammatory cytokinesJeong and Lee, 2011; Kawai and Akira, 2009). Here, we pro-ided evidence showing the potential inhibitory of Cha Fex on theoly(I:C)-induced IRF3 and NF-�B pathways in DCs through inhibit-

ng the phosphorylation of IRF3 and I�B-�. Furthermore, there arehree MAPK components: ERK1/2, JNK and p38, representing three

ajor mammalian MAPK cascades identified in intracellular sig-al transduction pathways. Unlike ERK1/2 which is responsible

or the induction of cellular processes, including mainly prolifer-tion and differentiation, JNK and p38 cascades are found to acts key components in the regulation of immunological effects.his may be the reason why Cha Fex could inhibit the phospho-ylation of JNK and p38 in poly(I:C)-induced DCs, but not havehe same effect on ERK1/2 (Brown et al., 2011; Plotnikov et al.,011).

In summary, our present study demonstrates that that Chaex can inhibit the maturation and function of poly(I:C)-inducedCs and exhibit an immunosuppressive effect on mouse bonearrow-derived DCs via TLR3 signaling, which suggests potential

pplication of Cha Fex in the treatment of autoimmune inflamma-ory diseases.

cknowledgements

This work was supported by the National Natural Science

oundation of China (90813036, 81072410, 81121062), the Scien-ific Research Foundation of Graduate School of Jiangsu ProvinceCXZZ11 0036), and the Scientific Research Foundation of Graduatechool of Nanjing University (2010CL04).

logy 51 (2012) 150– 158 157

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