International Immunopharmacology 9 (2009) 886–893

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International Immunopharmacology

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Surfactin inhibits immunostimulatory function of macrophages through blockingNK-κB, MAPK and Akt pathway

Sun Young Park, YoungHee Kim ⁎

Department of Molecular Biology, College of Natural Sciences, Pusan National University, Jangjeon-dong, Keumjeong-gu, Pusan, 609-735, Republic of Korea

⁎ Corresponding author. Tel.: +82 51 510 2526; fax: +E-mail address: yheekim@pusan.ac.kr (Y. Kim).

1567-5769/$ – see front matter © 2009 Elsevier B.V. Adoi:10.1016/j.intimp.2009.03.013

a b s t r a c t

a r t i c l e i n f o

Article history:Received 30 January 2009Received in revised form 20 March 2009Accepted 23 March 2009

Keywords:SurfactinAntigen presentationCostimulatory moleculesMajor histocompatibility complex (MHC)NF-kappaB (NF-κB)

Surfactin is one of the most powerful biosurfactants, and is known to have antibiotic, anti-tumor and anti-inflammatory functions. In this study, we investigated the effect of surfactin on antigen-presenting propertyof macrophages. Thioglycollate-elicited mouse peritoneal macrophages were tested for surface moleculeexpression, cytokine production, phagocytosis, capacity to induce T cell activation by mixed lymphocytereaction, and underlying signaling pathways. Surfactin significantly suppressed lipopolysaccharide-inducedexpression of CD40, CD54, CD80, and MHC-II, but not of CD86 and MHC-I. Surfactin-treated macrophages alsoexhibited impaired phagocytosis and reduced IL-12 expression. And surfactin markedly inhibited theactivation of CD4+ T cells. Impaired translocation and activation of NF-κB p65 were founded on macrophagesexposed to surfactin. In addition, surfactin inhibited the phosphorylation and degradation of IκB-α, andsuppressed the activation of IKK, Akt, JNK and p38 kinase. These results suggest that surfactin impair theantigen-presenting function of macrophages by inhibiting the expression of MHC-II and costimulatorymolecules via suppression of NF-κB, p38, JNK and Akt. These novel findings provide new insight into theimmunopharmacological role of surfactin in autoimmune disease and transplantation.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Macrophages are notmerely essential for innate immune response,but play a role in the activation of the adaptive immune system byacting as professional antigen-presenting cells (APCs) [1]. Activatedmacrophages express major histocompatibility complex-II (MHC-II)molecules and costimulatory and adhesionmolecules like CD80 (B7.1),CD86 (B7.2), CD40 and CD54 and induce an effective T cell response inthe presence of an antigen-dependent inflammatory response [2–6].Effective Tcell activation bymacrophages requiresMHC/Tcell receptorinteraction and costimulatory molecules on T cells and macrophages,which is supplemented by secretion of the T cell stimulatory cytokines(IL-12 and IL-18) by macrophages [7]. In the absence of costimulatorymolecules on APCs, antigen recognition by the T cell receptor may leadto anergy. In vivo studies using anti-CD80 and anti-CD86 monoclonalantibodies or geneticallymanipulated animals havedemonstrated thata blockade of costimulatory molecules can prolong allograft survivaland reduce the severity of autoimmune diseases [8,9]. Therefore,pharmacological intervention in the up-regulation of costimulatorymolecules may be useful in the treatment of autoimmune diseases orin transplantation [10–15].

82 51 513 3526.

ll rights reserved.

Macrophages, and other phagocytic cells such as neutrophils anddendritic cells (DCs), can discriminate between pathogens and selfthrough the signals triggered by pattern-recognition receptors (PRRs)present at the cell membrane. PRRs such as Toll-like receptors (TLRs)recognize different pathogens components referred to as pathogen-associated molecular patterns (PAMP). Lipopolysaccharide (LPS), anintegral element present at the outer membrane of Gram-negativebacteria, is recognized by TLR4. Interaction between TLR4 and its lig-and LPS initiates a complex signaling pathway including activation ofthe phosphatidylinositol-3-kinase (PI-3K) and Akt (protein kinase B),mitogen-activated protein kinases (MAPKs), inhibitory κB (IκB), IκBkinase (IKK),whichfinally leads to the activationof several transcriptionfactors that control macrophage activation such as nuclear factor-kappaB (NF-κB), interferon regulatory factor (IRF)-3, andAP-1 [16,17]. Of thesetranscription factors, NF-κB has been shown to play a major role inefficient antigen presentation and the expression of MHC and costi-mulatorymolecules [18,19]. Inunstimulatedcells, NF-κB is present in thecytosol bound to the inhibitory protein kappa B (IκB). In response tostimulation such as LPS, IκBs are rapidly phosphorylated by IκB kinases(IKKs) and then ubiquitinated and degraded by 26S proteasome com-plex. The freeNF-κB dimers translocate to the nucleus, bind to the kappaB motif of the target genes and stimulate their transcription.

Surfactin, a biosurfactant produced by Bacillus subtilis, is a cycliclipopeptide built from a heptapeptide and a β-hydroxy fatty acid withvariable chain lengths of 13–15 carbon atoms [20]. This biosurfactantis biodegradable and less toxic than chemical surfactants and has a lot

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of applicable potential in various fields including biomedicine [20].Surfactin has been reported to possess anti-viral [21] and anti-tumoractivities [22,23], inhibit fibrin clot formation and platelet aggrea-tion [24], and have an anti-inflammatory effect through inhibitionof platelet and spleen cytosolic phopholipase A2 (PLA2) [25] andsuppression of nitric oxide (NO) synthesis [26-28]. However, littleis known about the effect of surfactin on APCs, which are crucial inthe immune response. In this study, we investigated the effects ofsurfactin on antigen-presenting activities of macrophages and theexpression of MHC and costimulatory molecules. The activities ofNF-κB, and PI-3K/Akt and MAPK signaling pathway were alsoobserved.

2. Materials and methods

2.1. Reagents

Surfactin (from B. subtilis), LPS (phenol extracted from Salmonellaenteritidis), ovalbumin (OVA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and other reagents not refer-red were purchased from Sigma-Aldrich (St. Louis, MO). SB203580,SP600125 and LY294002 were purchased from A.G. Scientific(San Diego, CA). Mouse monoclonal antibodies against CD40, CD54,CD80, CD86, MHC-I (H-2Db) and MHC-II (I-Ab) were purchased fromBD Pharmingen (San Diego, CA, USA).

2.2. Macrophage culture

C57BL/6 (H-2Db and I-Ab) mice purchased from Dae Han Labor-atory Animal Center (Korea) were used between 8 and 12 weeksof age (25–30 g). They were housed in a specific pathogen-freeenvironment in our animal facility for at least 1 week before use.Thioglycollate (TG) broth (Brewer, Difco, Detroit, MI)-elicited macro-phages were harvested 3 days after intraperitoneal injection of 2.5 mlTG into mice and isolated as reported previously [29]. RAW 264.7 cellswere maintained in Dulbecco's modified Eagle's medium supple-mented with 10% FBS.

2.3. Cell viability assay

The cytotoxicity of surfactin was assessed using the microculturetetrazolium (MTT)-based colorimetric assay. TG-elicited mouseperitoneal macrophages were treated with various concentrations ofsurfactin in the absence or presence of LPS (1 µg/ml) for 24 h. MTTsolution (final concentration 62.5 µg/ml) was added to each well.After incubation for 3 h at 37 °C and 5% CO2, the supernatant wasremoved and the formed formazan crystals in viable cells weresolubilized with 150 µl of DMSO. Then the absorbance of each wellwas read at 570 nm using a microplate reader.

2.4. Flow cytometry analysis

TG-elicited mouse peritoneal macrophages were pre-treated withsurfactin for 2 h and cultured for 24 h in the absence (vehicle solution)or presence of LPS. Macrophages were harvested for staining withFITC-labeled monoclonal antibodies against CD40, CD54, CD80, CD86,MHC-I and MHC-II for 30 min, washed with PBS, and analyzed using aflow cytometer (Beckman Coulter, Miami, FL). A total of 10,000 livecell events as gated on forward and side scatter characteristics wereacquired.

2.5. Phagocytosis assay

Phagocytosis was determined using pHrodo™ E. coli BioParticles®

Phagocytosis Kit (Invitrogen, Paisley, UK). The assay was performedaccording to the manufacturer's instructions. Briefly, TG-elicited

mouse peritoneal macrophages were incubated with surfactin for2 h and then treated with LPS (1 µg/ml) for 24 h. Then, macrophageswere treated with BioParticles for 30 min at 37 °C in a humidified 5%CO2 incubator, washed three times in ice-cold PBS and analyzed byflow cytometry (Beckman Coulter).

2.6. Detection of IL-12p70 production by enzyme-linked immunosorbantassay (ELISA)

TG-elicited mouse peritoneal macrophages were incubated withvarious concentrations of surfactin for 2 h prior to treatment with LPS(1 µg/ml) for 24 h. IL-12p70 levels were quantified in culture mediaby ELISA Kit (Koma Biotech, Seoul, Korea) according to the manu-facturer's instructions.

2.7. Mixed lymphocyte reactions (MLRs)

CD3+ Tcells were purified from the spleens of C57BL/6micewith amouse T cell enrichment kit (StemCell Technologies, Vancouver, BC,Canada) by immunomagnetic beads according to the manufacturer'sinstructions. TG-elicited mouse peritoneal macrophages were treatedwith surfactin or vehicle solutions in the presence of 1 µg/ml LPS and500 µg/ml OVA for 24 h. Macrophages were washed with mediumthree times and specific numbers of macrophages (4×104 cells) ineach vehicle or surfactin-treated group were used for coculture with Tcells (2×105 cells) for 4 days. T cells were harvested for staining withFITC-labeled monoclonal antibodies against CD4 for 30 min, washedwith PBS and analyzed for CD4 fluorescence intensity by flow cyto-metry (Beckman Coulter).

2.8. Transient transfection and dual-luciferase assay

RAW 264.7 cells were transfected with κB-luc reporter plasmidconsisted of three κB concatamers from the Igγ chain and fireflyluciferase gene [29] using FuGENE-6 reagent (Roche Applied Science,Basel, Switzerland) according to the manufacturer's instructions.Renilla luciferase control plasmid pRL-CMV (Promega Madison, WI)was cotransfected as an internal control for transfection efficiency.Twenty four hours after transfection, the cells were incubated withsurfactin for 2 h and then treated with LPS (1 µg/ml) for 24 h.Luciferase activity was assayed with the dual-luciferase assay kit(Promega) according to the manufacturer's instructions. Lumines-cence was measured with GloMax™ 96 microplate luminometer(Promega).

2.9. Western blot analysis

The cells were harvested on ice-cold lysis buffer (1% Triton X-100,1% deoxycholate, 0.1% SDS). Protein content of cell lysates wasdetermined with Bradford reagent (Bio-Rad, Hercules, CA) and equalamounts of protein were separated electrophoretically using 10%SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophor-esis), and then the separated proteins were electrically transferred to0.45 µmnitrocellulose paper. The blot was incubatedwith anti-NF-κBp65, IκB-α, IKKα/β, p-IKKα/β, JNK, p38, (Santa Cruz Biotechnology,Santa Cruz, CA), p-IκB-α, p-JNK, p-p38, p-Akt, Akt (Cell SignalingTechnology, Danvers, MA) or α-tubulin antibody (Bio Genex, SanRamon, CA) and secondary antibody and then detected by the en-hanced chemiluminescence detection system according to therecommended procedure (Amersham, Piscataway, NJ).

2.10. Immunofluorescence confocal microscopy

TG-elicited mouse peritoneal macrophages were cultured direct-ly on glass cover-slips in a 35 mm dish. Cells were fixed with 3.5%paraformaldehyde in PBS for 10 min at room temperature and

Table 1Surfactin markedly inhibits the expression of costimulatory molecules and MHC-II onLPS-stimulated macrophages.

% positive cellsa

Surface maker Control Surfactin LPS Surfactin + LPS

CD40 19.4±3.9 20.1±5.2 69.4±7.3 38.6±9.0*CD54 14.7±2.8 16.3±4.5 66.7±5.1 44.7±3.0 **CD80 25.0±2.7 23.8±2.8 64.9±5.6 51.0±2.3 *CD86 21.0±1.9 22.3±2.6 46.7±9.1 42.7±8.2MHC-I 16.9±3.0 18.9±0.6 35.7±8.2 32.6±7.2MHC-II 18.8±0.9 20.4±1.4 60.0±2.0 32.9±3.6**

TG-elicited mouse peritoneal macrophages were cultured in the absence or presence ofsurfactin (30 µM) following the LPS (1 µg/ml) stimulation for 24 h. The expression ofsurface molecules was analyzed by FACS (Beckman coulter). *, **, The statisticalsignificance between samples with and without surfactin is indicated (*, pb0.05; **,pb0.01 vs LPS-stimulated macrophages).

a Values are means±S.E. of three independent experiments.

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permeabilized with 100% methanol for 10 min. To investigate thecellular localization of NF-κB, cells were treated with a polyclonalantibody against NF-κB p65 for 1.5 h. After extensive washing withPBS, cells were further incubated with a secondary FITC-conjugatedanti-rabbit IgG antibody for 1 h at room temperature. Nuclei werestained with 1 µg/ml of 4′,6-diamidino-2-phenylindole (DAPI) andanalyzed by confocal microscopy using a Zeiss LSM 510 Metamicroscope.

Fig. 1. Effect of surfactin on the expression of costimulatory molecules and MHC in plasma30 µM of surfactin (S) for 2 h, and then stimulated with 1 µg/ml of LPS for 24 h at 37 °C. Cellsusing flow cytometry. Histograms show profiles of isotype controls (gray) and monoclonal asurfactin on cell viability. Mouse peritoneal macrophages were treated with indicated conceviabilities were determined by MTT assay. Values are means±S.E. of three independent ex

2.11. Statistical analysis

All results are expressed as means±S.E. Each experiment wasrepeated at least three times. Statistical significances were com-pared between each treated group and analyzed by the PairedStudent's t-test. Data with pb0.05 were considered statisticallysignificant.

3. Results

3.1. Surfactin inhibits LPS-induced expression of MHC-II and costimulatorymolecules in macrophages

Macrophages, upon activation, are known to increase their surfaceexpression of MHC and costimulatory molecules involved in antigenpresentation. We observed that LPS-induced activation resulted inincreased expression of various surfacemolecules such as CD40, CD54,CD80, CD86, MHC-I, and MHC-II by mouse peritoneal macrophages.Surfactin inhibited LPS-induced surface expression of CD40, CD54,CD80 and MHC-II. Interestingly, little change in CD86 and MHC-Imolecules was observed (Table 1 and Fig. 1A). These results suggestthat surfactin might impair the antigen-presenting ability of macro-phages by suppressing the expression of MHC-II and costimulatorymolecules in the plasma membrane of macrophages.

To confirm that the inhibitory activity of surfactin was not due tothe direct cytotoxicity of surfactin on macrophages, we performed

membrane of macrophages. (A) Mouse peritoneal macrophages were pre-treated withwere harvested for staining with FITC-conjugated monoclonal antibodies and analyzedntibodies (black). The results are from one experiment of three performed. (B) Effect ofntration of surfactin in the absence or presence of LPS (1 µg/ml) for 24 h at 37 °C. Cellperiments. ⁎ pb0.05 and ⁎⁎ pb0.01 vs LPS-only group.

Fig. 2. Phagocytic activity of LPS-stimulated macrophages is suppressed by surfactin.(A) Mouse peritoneal macrophages were pre-treatedwith 30 µM of surfactin (S) for 2 h,and then stimulatedwith 1 µg/ml of LPS for 24 h. Phagocytic activitywas determined byflow cytometry using pHrodo™-conjugated bioparticles. (B) The percentage of marker-positive cells. Values are means±S.E. of three independent experiments. * pb0.01 vsLPS-only group.

Fig. 3. Inhibition of IL-12p70 production by surfactin.Mouse peritonealmacrophageswereincubatedwith various concentrations of surfactin (S) for 2 h, and then LPS (1 µg/ml)wastreated for 24 h. IL-12p70 in the cultured supernatant were measured by ELISA kit. Valuesare means±S.E. of three independent experiments. ⁎ pb0.05 and ⁎⁎ pb0.01 vs LPS-onlygroup.

Fig. 4. Antigen-presenting activity of macrophages to CD4+ T cell is suppressed bysurfactin. (A) Mouse peritoneal macrophages were treated with 1 µg/ml LPS and500 µg/ml ovalbumin for 24 h in the absence or presence of surfactin (S). Cells werewashed and cocultured with T cells purified from C57BL/6 mice splenocytes at ratios of1:5 for 4 days. T cells were harvested for staining with FITC-conjugated monoclonalantibodies against CD4 and analyzed by flow cytometry. (B) The percentage of CD4-positive cells. Values are means±S.E. of three independent experiments. * pb0.01 vsLPS-only group.

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MTT-based cell viability assays. Mouse peritoneal macrophages werepre-treated with various concentrations of surfactin for 2 h, and thenstimulated with 1 µg/ml of LPS for 24 h. As shown in Fig. 1B, cellviability was not reduced by surfactin. Instead, surfactin protectedcells from LPS cytotoxicity in a dose-dependent manner. Surfactinalone had no effect on the viability of macrophages. Therefore, ourdata were not the result of non-specific effects due to the cytotoxicityof surfactin.

3.2. LPS-induced phagocytic activity of macrophages is inhibited bysurfactin

Macrophages act as APCs to produce antigen molecules on the cellsurface after phagocytosis, the incorporation and digestion of anti-genic molecules. So we studied the effect of surfactin on bacterialphagocytosis by activated macrophages. Phagocytotic activity ofmouse peritoneal macrophages was assessed in terms of theirincorporation of fluorescent E. coli particles. As shown in Fig. 2,surfactin inhibited LPS-induced phagocytotic activity of macrophages.Phagocytotic activity of macrophages treated with surfactin alone(18.1%±3.3%) was similar to that of control.

3.3. Surfactin suppresses the secretion of IL-12 by macrophages

IL-12 is produced by activated macrophages and plays a centralrole in driving the development of Th1 immune responses. Weexamined the effect of surfactin on IL-12p70 secretion of macro-phages. IL-12p70-specific ELISAwas used to detect the release of IL-12in the culture medium of macrophages. As shown in Fig. 3, IL-12secretion was inhibited by surfactin in a dose-dependent mannercomparable to that observed upon treatment with LPS alone,while IL-12 level was not affected by treatment with surfactin alone(69.6±9.2 pg/ml). This result suggests that surfactin suppressesT cell activation through inhibiting the production of IL-12, a Th1-inducing cytokine, in macrophages.

3.4. Surfactin inhibits the ability of macrophages to stimulate T cells

We next determined whether surfactin could affect the ability ofmacrophages to stimulate T cells by use of mixed lymphocyte re-actions (MLR). Mouse peritoneal macrophages were pre-stimulatedwith LPS and OVA in the absence or presence of surfactin for 24 h,and cocultured with CD3+ T cells (macrophage:T cell ratios of 1:5)for 4 days. And then T cells were stained with FITC-labeled mono-clonal antibodies against CD4 and analyzed for CD4 fluorescenceintensity by flow cytometry. As shown in Fig. 4, surfactin signifi-cantly inhibited CD4+ T cell activation while LPS-stimulated macro-phages activated CD4+ T cells. Macrophages treated with surfactin

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alone showed little effect (percentage of CD4+ T cells was 29.4%±2.6%). When total T cell proliferation was estimated, a similar resultwas obtained; LPS-treated group was 1.8-fold and LPS and surfactin-cotreated group was 1.3-fold that of the control group (data not

Fig. 5. Effect of surfactin on LPS-induced NF-κB activity and the phosphorylation of Akt, p38 amacrophage cells were cotransfected with κB-luc reporter and control renilla luciferase plasmof surfactin for 2 h, and then stimulated with LPS (1 µg/ml) for 24 h. Equal amounts of cell excontrol renilla luciferase expression. All transfection experiments were performed in duplicavs LPS-only group. (B) Effect of surfactin on nuclear translocation of NF-κB. Mouse peritoneastimulated with LPS (1 µg/ml) for 30 min. Nuclear and cytosolic proteins were extracted, adetection of TATA-binding protein (TBP) and α-tubulin estimated protein-loading control fowere measured by densitometer and indicated as a fold to control. (C) Nuclear translocationincubated as described above on cover slip in 6 well plates. Fixed cells were stained with DAwere taken by confocal microscopy (bar is 10 µm). (D) Mouse peritoneal macrophages were(1 µg/ml) for 15 min. Cells were harvested and equal amount of cytosolic extracts were analycontrol for each lane. (E) Mouse peritoneal macrophages were treated with indicated conceand SP600125 for JNK; 20 µM, respectively) for 2 h and stimulated with LPS (1 µg/ml) for

shown). Our results suggest that surfactin impairs the antigen pre-sentation of macrophages through suppressed expression of MHC-IIand costimulatory molecules, inhibiting phagocytosis and blockingIL-12 secretion by macrophages.

nd JNK in macrophages. (A) Effect of surfactin (S) on NF-κB transactivation. RAW 264.7id pRL-CMV. Twenty four hours later, cells were incubated with various concentrationstracts were assayed for dual-luciferase activity. κB luciferase activity was normalized tote in three independent experiments. Data are expressed as the means±S.E. ⁎⁎ pb0.01l macrophages were incubated with various concentrations of surfactin for 2 h, and thennd then Western blotting was conducted with anti-NF-κB p65 antibody. Western blotr each lane. Relative intensities of p65 protein bands compared to TBP or tubulin bandsof NF-κB was confirmed by confocal microscopy. Mouse peritoneal macrophages werePI and anti-NF-κB p65 antibody and FITC-conjugated anti-rabbit IgG antibody. Images

incubated with various concentrations of surfactin for 2 h, and then stimulated with LPSzed byWestern blotting. Western blot detection ofα-tubulin estimated protein-loadingntrations of surfactin or specific kinase inhibitors (LY294002 for Akt, SB203580 for p38,15 min. Equal amount of cell extracts was analyzed by Western blotting.

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3.5. Surfactin inhibits LPS-induced NF-κB activation in macrophages

Since NF-κB is known to play a critical role in antigenpresentation, weinvestigated whether surfactin inhibits NF-κB activation. RAW 264.7macrophage cells were transiently transfected with luciferase reportergenes driven by NF-κB binding consensus concatamers (κB-luc) and theeffect of surfactin on reporter gene expressionwas examined. As shown inFig. 5A, surfactin inhibited LPS-induced transactivation of NF-κB in a dose-dependentmanner, while luciferase activity of cells treatedwith surfactinalonewas 0.91±0.2 fold higher. SinceNF-κB translocates from the cytosolto the nucleus when it is activated, we examined the effect of surfactin onthe localization of NF-κB. Mouse peritoneal macrophages were treatedwith LPS in the presence or absence of surfactin, and cytosolic and nuclearextracts were analyzed by immunoblotting with anti-p65 antibody.Surfactin inhibited LPS-induced nuclear translocation of NF-κB p65 in adose-dependent manner (Fig. 5B). To confirm this result, the location ofNF-κBp65 subunitwasmeasuredbyconfocalmicroscopy. Consistentwithimmunoblotting, most p65 is stained in the cytosol of unstimulated cells,and LPS-stimulated cells showed co-staining of p65 and DAPI indicatingnuclear localization of p65, while nuclear accumulation of p65 wasdecreased by surfactin (Fig. 5C). The level of NF-κB p65 of the surfactinalone group in Western blotting and confocal microscopy was similar tothat of the control group (data not shown). These results indicate thatsurfactin inhibits LPS-induced activation of NF-κB.

To analyze whether surfactin affect NF-κB signaling pathway, weinvestigated the effects of surfactin on upstream signaling pathwayof NF-κB, that is, IκB-α phosphorylation and degradation. Mouseperitoneal macrophages were incubated with various concentrationsof surfactin for 2 h and stimulated with 1 µg/ml of LPS for 15 min. Asshown in Fig. 5D, surfactin significantly inhibited phosphorylationand degradation of IκB-α. At the same time, surfactin inhibitedphosphorylation of IKK, indicative of IIKK activity, in a dose-dependent manner, but the level of IKK protein was not changed bysurfactin. Surfactin alone did not affect the level of IκB-α, phospho-IκB-α and phospho-IKKα/β (data not shown). Therefore theseresults suggest that surfactin blocks IκB-α degradation throughinhibition of IκB-α phosphorylation by IKK.

3.6. Surfactin regulates PI-3K/Akt and MAPKs pathway in macrophages

It has been known that there is crosstalk betweenMAPKs, PI-3K/Aktand NF-κB, and that crosstalk is important for maximal activation ofsome genes [30–33]. So we examined the effect of surfactin on PI-3K/Akt and MAPK activities which regulate the function of NF-κB. Mouseperitoneal macrophages were treated with indicated concentrations ofsurfactin or specific inhibitors for 2 h and then stimulated with 1 µg/mlof LPS for 15min. Surfactin suppressed LPS-induced phosphorylation ofAkt, p38 and JNK in a dose-dependent manner (Fig. 5E). Surfactin alonedid not affect the level of phosphorylated kinases (data not shown). Theamount of non-phosphorylated Akt, p38 and JNK were unaffected byeither LPS or surfactin treatment. These data suggest that the inhibitoryeffect of surfactin on antigen presentation should be associated withsuppressed activation of p38, JNK and PI-3K/Akt in macrophages.

4. Discussion

Surfactin is a cyclic lipopeptide produced by B. subtilis. B. subtilis isa gram-positive bacterium, and a strain of B. subtilis formerly knownas Bacillus natto is used in the Japanese delicacy natto as well as in thesimilar Korean food Cheonggukjang (fermented soybean paste).Surfactin is one of the most powerful biosurfactants, possessing at-tractive therapeutic and biotechnological properties such as anti-tumor and anti-inflammatory activities. In the present study, wedemonstrate that surfactin can effectively down-regulate surfacemolecules (CD40, CD54, CD80, MHC-II) of activated macrophages aswell as the level of IL-12. We also demonstrate the inhibition of

phagocytosis and T cell activation by surfactin and underlying mole-cular mechanism. On the basis of cytotoxicity assay, we confirmedthat the concentrations we used in this study do not have directtoxicity on macrophages.

It has been well documented that macrophages can be subse-quently activated after treatment with PAMP such as LPS. AlthoughDCs are known to bemajor professional APCs, activatedmacrophagesact as professional APCs and exhibit the property to prime T cellsthrough the up-regulation of the MHC-I, and -II molecules, and thecostimulatory molecules CD80, CD86, CD40 and CD54 [2–4]. In thisstudy, surfactin inhibited the expression of MHC-II, CD80, CD40 andCD54 on the surface of LPS-stimulated macrophages. Recently,macrophages from CD54 knockout mice were shown to havedecreased phagocytic activity indicating an additional role of thismolecule in phagocytosis [34]. Thus these results suggest surfactinmight inhibit not only T cell activation but also phagocytic activitythrough the suppression of CD54 expression. The interaction of CD80or CD86 with CD28 on T cells increases the efficacy of signalingthrough the T cell receptor (TCR) and MHC/peptide complex, whichtightly correlates with T cell activation. CD28-mediated costimula-tion up-regulates bcl-xL and provides a critical anti-apoptotic signalthat protects Tcells from suicidal Fas-mediated apoptosis during TCR-mediated activation [35]. Therefore, blockade of CD80 or CD86/CD28costimulation could give rise to increased Fas-mediated apoptosisand suppress T cell activation, thus leading to T cell anergy.

The classical paradigm of antigen presentation by professionalAPCs is that endogenous antigens are presented via MHC-I moleculesto CD8+ Tcells, whereas exogenous antigens are presented via MHC-IImolecules to CD4+ T cells [36]. In addition to the processing andpresentation of antigenic peptides via MHC molecules, APCs play acentral role in driving CD4+ T helper cell differentiation through theproduction of critical cytokines such as IL-12 [37]. Additionally, it hasbeen suggested that CD80-binding stimulates a Th1 response, result-ing in the secretion of cytokines that promote cellular immune re-sponses including inflammation, whereas binding of CD86 evokes aTh2 response, which is characterized by anti-inflammatory and pro-humoral cytokines [10]. In the present study, we demonstrate thatsurfactin inhibited the up-regulation of MHC-II, but not of MHC-I, andactually impaired CD4+ T cell expansion in MLR. Surfactin also in-hibited the production of IL-12p70 and CD80, not of CD86, by macro-phages. All of these results suggest that surfactin suppresses MHC-II-restricted exogenous antigen presentation to CD4+ T cells. Moreover,surfactin might inhibit the Th1 response by suppressing IL-12p70secretion and CD80 expression, although whether surfactin impairsTh1 cytokine production in T cells from MLR remains to be clarified.The inhibition of Th1 development exerts negative regulatory roles fora wide variety of immune cells [38]. Hence, the surfactin-mediatedinhibition of IL-12 production from LPS-stimulated macrophagesmight also contribute to the induction of the immunosuppressivestate. Consistently, surfactin inhibited the expression of proinflam-matory cytokines such as IL-6 and IL-1β in LPS-stimulated macro-phages (our unpublished data and [26,27]). On the other hand, itmight be that surfactin suppress the maturation and antigen-pre-senting property of DCs, which are major professional APCs, althoughthe effect of surfactin on DCs remains to be examined.

Since several studies have demonstrated that NF-κB is requiredfor efficient antigen presentation [19], we investigated the effect ofsurfactin on NF-κB activation pathway in macrophages. Our datashowed that surfactin inhibited both NF-κB activity in a reporterassay and translocation from cytoplasm to nucleus. Phosphoryla-tion of IκB-α by the IκB kinases results in the ubiquitination andproteasomal degradation of IκB-α. This process, the key step in theregulation of the NF-κB pathway, releases the NF-κB proteins fromIκB-α, leading to their nuclear translocation and binding to κBpromoter elements to result in activation of gene expression. Wefound the level of IκB-α phosphorylation and degradation was

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decreased in surfactin-treated macrophages. And the phosphoryla-tion of IKK, indicative of IKK activation, was also decreased bysurfactin. These results indicate that surfactin-mediated decrease ofIKK and IκB phosphorylation reduces the down-stream activationand translocation of NF-κB, and may account for the inhibition ofthe antigen presentation by macrophages.

MAPKs have been shown to be involved in the LPS-mediatedinduction of many inflammatory genes and are important for theactivation of NF-κB. Previous studies have demonstrated that MAPKsare involved in LPS-induced surface molecule expression. For exam-ple, JNK mediates LPS-induced up-regulation of CD80, CD83, CD86,CD40 and CD54 [39], and p38 kinase is involved in LPS-induced CD80,CD83, CD86, CD40, CD54 and HLA-DR expression [40,41]. PI-3K andits down-stream kinase Akt pathway have been reported to beinvolved in maturation and antigen presentation in DCs [42].Furthermore, the crosstalk between MAPKs, PI-3K/Akt and NF-κB isimportant for maximal activation of some genes [30,32,33]. Thus, weexamined the effect of surfactin on MAPK and PI-3K/Akt pathway.We found that surfactin inhibited LPS-induced activity of JNK, p38kinase and Akt in macrophages. These results indicate that theinhibitory effect of surfactin on antigen presentation should beassociatedwith suppressed activation of MAPK, PI-3K/Akt and NF-κBas potential targets. Recently surfactin was shown to inhibit NOproduction by suppressing NF-κB activation in LPS-stimulatedmacrophages [28], which is consistent with our data. But Akt activitywas not affected by surfactin in their report. On the contrary, wefound that surfactin at the same condition as previously done [28]inhibited the phosphorylation of Akt in response to LPS in RAW264.7macrophage cells (data not shown).

In summary, this study shows that surfactin suppresses surfaceexpression of MHC-II and costimulatory molecules in macrophages,IL-12p70 production, phagocytosis of antigen and subsequent anti-gen presentation to CD4+ T cells through the inhibition of NF-κB, PI-3K/Akt, p38 kinase and JNK. These consistent results indicate surfactinis a potent immunosuppressive agent and suggest an importanttherapeutic implication for transplantation and autoimmune diseases,including arthritis, allergy, and diabetes.

Acknowledgment

This work was supported by the Korea Research Foundation Grantfunded by the Korean Government (MOEHRD, Basic Research Promo-tion Fund) (KRF-2005-204-C00050).

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