International Immunopharmacology 16 (2013) 79–84

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International Immunopharmacology

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Alkaloids fromGalipea longiflora Krausemodify thematuration of humandendritic cells and their ability to stimulate allogeneic CD4+ T cells

Jacqueline Calla-Magariños a,b,c,⁎, Carmen Fernández b, Marita Troye-Blomberg b, Jona Freysdottir a,d

a Department of Immunology and Center for Rheumatology Research, Landspitali, The National University Hospital of Iceland, Reykjavik, Icelandb Department of Immunology, The Wenner Gren Institute, Stockholm University, Stockholm, Swedenc Laboratory of Immunology, SELADIS Institute, Biochemistry and Pharmacy Faculty, Universidad Mayor de San Andrés, La Paz, Boliviad Faculty of Medicine, Biomedical Center, University of Iceland, Reykjavik, Iceland

⁎ Corresponding author at: Department of ImmunoloStockholm University, 106 91 Stockholm, Sweden.

E-mail address: jaqueline.calla@imun.su.se (J. Calla-

1567-5769/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.intimp.2013.03.022

a b s t r a c t

a r t i c l e i n f o

Article history:Received 26 September 2012Received in revised form 14 March 2013Accepted 15 March 2013Available online 2 April 2013

Keywords:LeishmaniasisGalipea longiflora KrauseNatural productsDendritic cellsCytokines

Alkaloids obtained from the plant Evanta have been shown to have dual effects in Leishmania infection; a di-rect leishmanicidal effect on the parasite and more importantly, the alkaloids affect both polyclonal andLeishmania-specific stimulation of T-cells.Dendritic cells (DCs) play a pivotal role in stimulation and polarization of naïve T cells towards a Th1, Th2,Th17 or regulatory phenotype. In leishmaniasis, the interactions between the parasites and DCs are complexand involve contradictory functions that can stimulate or suppress T cell responses, leading to the control ofinfection or progression of disease.In this study the effect of an alkaloid extract of Evanta (AEE) or the purified alkaloid 2-phenilquinoline (2Ph)on the activation of human DCs and their ability to stimulate allogeneic CD4+ T cells was analyzed. The ex-pression of surface activation molecules was not affected on DCs stimulated in the presence of AEE or 2Phnor did AEE-DCs or 2Ph-CDs affect the expression of activation surface molecules on allogeneic CD4+

T cells. In contrast, as compared with control, the secretion of IL-12p40, IL-23 and IL-6 was lower fromAEE-DCs and 2Ph-CDs and allogeneic CD4+ T cells co-cultured with these DCs secreted lower levels ofIFN-γ and IL-10 but the same levels of IL-17.These results demonstrate that AEE and 2Ph affect the stimulation of DCs and their ability to stimulate allo-geneic CD4+ T cells by reducing the production of IFN-γ, IL-12 p40, IL-6 and IL-23. This suggests that AEE and2Ph may take part in regulation of inflammation.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

The plant Galipea longiflora Krause, locally known as Evanta, isused in Bolivia for the treatment of leishmaniasis and other parasiticdiseases [1–3]. Quinolinic alkaloids have been previously isolatedand characterized from different organs of this plant (bark, root andleaf) and all showed leishmanicidal effect [4,5].

Our previous studies on the in vitro activity of an alkaloid extractof Evanta (AEE) showed that, apart from the direct effect on the par-asites, pretreatment with AEE interfered with the proliferation andIFN-γ production of polyclonally activated T cells [6]. We also foundthat in vitro and in vivo AEE treatment interfered with restimulationof primed T cells, observed by reduction in the production of IFN-γ,IL-12 and tumor necrosis factor (TNF). In addition, we showed thattreatment of mice infected with Leishmania braziliensis with AEEchanged the dynamics of the infection and controlled the footpathswelling and the parasite load [7]. These characteristics might be of

gy, The Wenner Gren Institute,

Magariños).

rights reserved.

benefit to avoid the inflammatory reaction and the tissue destructionseen in Leishmania infection [8–11].

As the immune system plays a role in the etiology or the patho-physiology of many diseases, such as infections and many chronic in-flammatory diseases, modulation of the immune response has beenan important goal to achieve. Increasing interest has been on identify-ing plant-derived natural substances with immunomodulatory capac-ity to control the outcome of certain immune responses with thenotion that their immunomodulatory properties can be used as alter-native or supplement to conventional medications that often have ad-verse side effects.

Dendritic cells (DCs) have a central role in initiating an immune re-sponse by acting as bridge between the innate and the acquired re-sponses and can induce both humoral and cell-mediated responses[12]. DCs play an important role in T cell stimulation and the polariza-tion of naïve T cells towards a Th1, Th2, Th17 or regulatory phenotype,as determined by the expression of different transcription factors andsecretion of different cytokines. The polarization of T cells dependsmainly on the dominant type of cytokines and costimulatory moleculesprovided by DCs [13,14], which in turn is dependent on the type anddose of the antigen present during their activation [15].

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In leishmaniasis, the interactions between the parasites and DCsare complex and involve contradictory functions that can stimulateor suppress T cell responses, leading to the control of infection or pro-gression of disease [16–18]. The activation of DCs in Leishmania infec-tions might be influenced by the different species/strains anddevelopmental stages of the parasite, which subsequently affect thesecretion of cytokines and expression of costimulatory molecules byDCs, which in turn, affect the T cell stimulation and T helper (Th)cell differentiation and hence the outcome of the infection [19].

The aim of this study was to investigate whether and how AEEcould affect the stimulation of human monocyte-derived DCs interms of expression of surface markers or production of cytokinesand to analyze whether AEE-treated DCs could affect allogeneic Tcell stimulation and polarization.

2. Materials and methods

2.1. Plant extract

AEE was isolated from the bark of the plant G. longiflora Krause(collected in the North of La Paz, Bolivia) by fractionation and purifica-tion, monitored by bioassay, as previously described [6]. The alkaloidmost frequently found in the extract, 2-phenylquinoline (2Ph), waspurified from the total extract [20]. Both AEE and 2Ph were kindlyprovided by A Giménez, IIFB Institute, Biochemistry and PharmacyFaculty, Universidad Mayor de San Andrés, La Paz, Bolivia. In thisstudy, AEE and 2Ph were dissolved in 0.1% dimethylsulfoxide(DMSO) (Sigma-Aldrich, Stockholm, Sweden). The DMSO used atthis concentration showed no interference with the parameters mea-sured and it was used as a control in all the experiments performed.

2.2. Reference drug

Meglumine antimoniate (glucantime, Sanofi-Aventis, Brazil) wasused as a drug of reference. It is the conventional drug used to treatleishmaniasis and is represented by its content of pentavalentantimonium (SbV).

2.3. Culture conditions

The cells were cultured in complete RPMI-1640 medium(Invitrogen, England) containing 2 mM L-glutamine and supplementedwith 10% heat inactivated fetal calf serum (FCS) (Invitrogen), 100 U/mlpenicillin, and 100 μg/ml streptomycin (Invitrogen), at 37 °C and 5 %CO2.

2.4. Generation of monocyte derived CDs

Monocyte-derived dendritic cells were obtained as previously de-scribed [21]. In brief, peripheral blood mononuclear cells (PBMCs)from human healthy donors (approved by the National BioethicsCommittee, Iceland) were obtained by density-gradient centrifuga-tion using Ficoll Histopaque (Sigma-Aldrich, Germany). CD14+

monocytes were isolated using CD14-microbeads (Miltenyi Biotec,Germany) with purity over 95% as determined by flow cytometry.In order to obtain DCs, CD14+ monocytes were cultured in 48-wellculture plates (Nunc, Denmark) at 5 × 105 cells/ml in the presenceof 12.5 ng/ml of IL-4 and 25 ng/ml of GM-CSF (both from R&DSystems, England) for seven days, with fresh media and cytokinesadded at day 3 or 4. After seven days the DCs were harvested and an-alyzed for the expression of surface molecules and supernatants werecollected for cytokine measurement.

2.5. Activation of DCs

In order to activate the DCs, cells were cultured in 48-well cultureplates at 2.5 × 105 cells/ml for 48 h at 37 °C, 5 % CO2, with IL-1βat 10 ng/ml, TNF at 50 ng/ml (both from R&D Systems) and lipopoly-saccharides (LPS) at 500 ng/ml (Sigma-Aldrich). AEE at 0.1, 1, and10 μg/ml, 2Ph at 0.1, 1, and 10 μg/ml, SbV at 28 μg/ml, AEE at 10 μg/mlplus SbV at 28 μg/ml SbV/ml (AEE/SbV) and vitamin D3 at4 × 10−11 mol/ml (Biomol International, USA) were added tosome wells. As AEE and 2Ph were dissolved in DMSO, cells werealso cultured in the presence of 0.1% DMSO (control). After 48 hthe DCs were harvested and analyzed for the expression of surfacemolecules and supernatants collected for cytokine measurement.

2.6. Co-culture of activated DCs with allogeneic CD4+ T cells

Allogeneic CD4+ T cells were obtained from PBMCs using CD4+

microbeads (Miltenyi Biotec) with the purity over 95% as determinedby flow cytometry. The CD4+ T cells at 2 × 106 cell/ml were co-cultured with stimulated DCs at 2 × 105 cell/ml in 96-well round bot-tomed culture plates (Nunc) for 6 days. The DCs had been matured inthe presence of AEE at 10 μg/ml (AEE-DCs), 2Ph at 10 μg/ml(2Ph-DCs) or 0.1 % DMSO (DMSO-DCs). Thereafter cells were harvestedand analyzed for the expression of surface molecules and supernatantcollected for cytokine measurement.

2.7. Phenotypic analyses of CDs and T cells by flow cytometry

Flow cytometric analysis was performed to determine the purity ofthe isolated cells, the differentiation of monocytes into DCs and activa-tion of DCs and T cells. Fluorochrome-conjugated monoclonal antibod-ies against CD86 and HLA-DR (AbD Serotec, England; BD Bioscience,USA) were used to stain monocytes and DCs; and against CD3, CD4,CD40L, CD62L, CD54, and CD69 (AbD Serotec; BD Bioscience) to stainthe T cells. Cells stained with fluorochrome-conjugated isotype controlantibodies were used as controls. The cells were analyzed using a flowcytometer (FACS Calibur, BD Bioscience) and the distribution of thecell population analyzed using CellQuest Pro acquisition software (BDBiosciences). The results were expressed as percentage of positivecells and as mean fluorescence intensity (MFI).

2.8. Cytokine detection

The levels of IL-6, IL-10, IL-12p40, IL-17, IL-23 and IFN-γ in culturesupernatants were determined using DuoSet ELISA (R&D Systems)according to the manufacturer's instructions. Results were expressedas pg/ml.

2.9. Statistical analysis

Data are presented as themean value ± standard error of themean(SEM). One-way ANOVA test and Tukey's post-hoc test were used todetermine statistical significant differences between treatments(GraphPad Prism, GraphPad software, USA; SigmaStat, Systat Software,USA). Differences were considered significant when p b 0.05.

3. Results

3.1. Stimulation of CDs in the presence of AEE did not modify the expres-sion of surface markers but decreased the production of IL-12

In this study we examined whether AEE or 2Ph could interferewith the in vitro maturation of monocyte-derived DCs. The DCswere stimulated for 2 days with IL-1β, TNF and LPS in the presenceof AEE at 0.1, 1 and 10 μg/ml; 2Ph at 0.1, 1 and 10 μg/ml; SbV at28 μg/ml, alone (SbV) and in combination with AEE at 10 μg/ml

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(AEE/SbV); or vitamin D3 at 4 × 10−11 mol/ml. Cells stimulated inthe presence of DMSO were used as controls.

DCs were analyzed by light microscopy after stimulation andshowed the characteristic activated DC features (being non-adherent,clustered and with protruding veils) regardless of treatment and thecell viability exceeded 97 % when tested by trypan blue (data notshown). Stimulating the DCs in the presence of AEE, 2Ph, SbV, AEE/SbVor vitamin D3 had no effect on the expression of the costimulatorymol-ecules CD86 and HLA-DR, neither the percentage of positive cells northe MFI, as compared with stimulated in the presence of DMSO(Fig. 1a and b).

DCs stimulated in the presence of AEE at 10 and 1 μg/ml, 2Ph at10 μg/ml, vitamin D3 or AEE/SbV secreted lower amounts ofIL-12p40 as compared with DCs stimulated in the presence of DMSO(Fig. 2a). On the contrary, the secretion of IL-10 was not significantlymodulated by any of the test compounds (Fig. 2b). The secretion ofIL-23 and IL-6 was analyzed after stimulation of the DCs in the pres-ence of AEE and 2Ph at 10 μg/ml and the IL-23 secretion was signifi-cantly reduced compared with DCs stimulated in the presence of

Fig. 1. Effects of AEE on immune stimulation of DCs measured by the expression ofco-stimulatory molecules. Human monocyte-derived DCs were stimulated with IL-1β(10 ng/ml), TNF (50 ng/ml) and LPS (500 ng/ml), in the presence of AEE at 0.1, 1,and 10 μg/ml, 2Ph at 0.1, 1, and 10 μg/ml, SbV at 28 μg/ml either alone or with AEEat 10 μg/ml (AEE/SbV), vitamin D3 at 4 × 10−11 mol/ml or 0.1% DMSO for 2 days.The expression of co-stimulatory molecules (CD86 and HLA-DR) was analyzed byflow cytometry and the results expressed as the mean ± SEM of percentage of positivecells (a) and mean fluorescence intensity (MFI (b)).

Fig. 2. Effects of AEE and 2Ph on stimulation of DCs measured by the secretion ofIL-12p40 and IL-10. Human monocyte-derived DCs were stimulated with IL-1β(10 ng/ml), TNF (50 ng/ml) and LPS (500 ng/ml), in the presence of AEE at 0.1, 1,and 10 μg/ml, 2Ph at 0.1, 1, and 10 μg/ml, SbV 28 μg/ml either alone or with AEE at10 μg/ml (AEE/SbV), vitamin D3 at 4 × 10−11 mol/ml or 0.1% DMSO for 2 days. Super-natants were collected and the levels of IL-12p40 (a) and IL-10 (b) secretion weredetected by ELISA with data given as the mean ± SEM. *** indicate statistical differ-ence (p b 0.001) between DCs stimulated in the presence of test compounds as com-pared with DCs stimulated in the presence of DMSO (one way ANOVA).

DMSO (Fig. 3b) and there was a trend for reduced secretion of IL-6although the difference was not significant (Fig. 3a).

3.2. Stimulating DCs in the presence of AEE affected their capacity tostimulate allogeneic CD4+ T cells

The ability of DCs stimulated in the presence of AEE or 2Ph to ac-tivated CD4+ T cells was investigated. DCs were stimulated in thepresence of 10 μg/ml of AEE (AEE-DCs), 10 μg/ml of 2Ph (2Ph-DCs)or DMSO (DMSO-DCs). After 48 h incubation the DCs wereco-cultured with allogeneic CD4+ T cells and incubated for further6 days.

Analysis of surface markers on the T cells showed no effect ofco-culture with AEE-DCs or 2Ph-DCs on the expression of CD3,CD40L, CD54, CD62L or CD69 as compared with cells co-culturedwith DMSO-DCs (data not shown).

Allogeneic CD4+ T cells that were co-cultured with AEE-DCs or2Ph-DCs secreted substantially less amount of IFN-γ (Fig. 4a) andIL-10 (Fig. 4b) than allogeneic CD4+ T cells co-cultured with DMSO-DCs,whereas no statistically significant effect was observed on IL-17 se-cretion by the allogeneic CD4+ T cells, although a trend for reduced

Fig. 3. Effects of AEE and 2Ph on stimulation of DCs measured by IL-6 and IL-23 secretion.Human monocyte-derived DCs were stimulated with IL-1β (10 ng/ml), TNF (50 ng/ml)and LPS (500 ng/ml), in the presence of AEE at 10 μg/ml, 2Ph at 10 μg/ml or 0.1% DMSOfor 2 days. Supernatants were collected and the levels of IL-6 (a) and IL-23 (b) secretionin DCs were measured by ELISA with data given as the mean ± SEM. p-Values indicatestatistical difference between DCs stimulated in the presence of AEE or 2Ph as comparedwith DCs stimulated in the presence of DMSO (one way ANOVA).

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secretion was observed (Fig. 4c). The cytokine secretion of the DCs atthe end of the co-culture was determined and we found that AEE-DCsand 2Ph-DCs secreted significantly less IL-6 and IL-23 than DMSO-DCs(Fig. 5a and b) and the secretion of IL-12p40 was slightly reducedalthough the difference was not statistically significant (Fig. 5c).

Fig. 4. Cytokine production of allogeneic CD4+ T cells activated by DCs stimulated inthe presence of AEE and 2Ph. Human monocyte-derived DCs were stimulated withIL-1β (10 ng/ml), TNF (50 ng/ml) and LPS (500 ng/ml) in the presence of AEE at10 μg/ml (AEE-DC), 2Ph at 10 μg/ml (2Ph-DC) or 0.1% DMSO (DMSO-DC) for 2 daysand subsequently co-cultured with allogeneic CD4+ T cells for 6 days. Supernatantswere collected and the levels of IFN-γ (a), IL-10 (b) and IL-17 (c) secretion were mea-sured by ELISA with data given as the mean ± SEM. p-Values indicate statistical differ-ence between DCs stimulated in the presence of AEE or 2Ph as compared with DCsstimulated in the presence of DMSO (one way ANOVA).

4. Discussion

Our previous studies showed that AEE could suppress the produc-tion of proinflammatory cytokines of activated lymphocytes [6,7].However, whether AEE affected only T cells or could also affect theability of DCs to stimulate the T cells, was not known. Therefore theinfluence of AEE and 2Ph on the stimulation of human monocyte-derived DCs and their ability to activate allogeneic CD4+ T cells wasanalyzed.

DCs stimulated in the presence of AEE or 2Ph produced lowerlevels of IL-12p40, an important Th1 inducing cytokine [22], andIL-23, a Th17 promoting cytokine [23]. It was observed that therewas a tendency to lower secretion of IL-6 a Th17 promoting cytokinewhich also mediated local inflammation [24]. However, secretion ofIL-10, an immunoregulatory cytokine, responsible for tolerogenicproperties of DCs [25,26], was not affected. These results are consis-tent with our previous results and indicate that AEE and 2Ph mayhave an anti-inflammatory effect by lowering the levels of thepro-inflammatory cytokines without affecting the anti-inflammatorycytokines [6,7].

Stimulation of DCs in the presence of AEE or 2Ph did not affect theexpression of the activationmarkers CD86 or HLA-DR, neither the per-centage of positive cells nor the mean expression levels. This indicatesthat AEE has no capacity to affect the expression of co-stimulatorymolecules of stimulated DCs and may be the explanation whyco-culturing the allogeneic CD4+ T cells with the DCs that had previ-ously been treated with AEE or 2Ph did not affect the expression ofvarious T cell activation markers, such as CD54, CD62L, CD69 andCD40L. This suggests that the CD4+ T cells were fully activated inthe presence of DCs, regardless of the treatment DCs received duringtheir stimulation.

Fig. 5. Cytokine production after co-culture of allogeneic CD4+ T cells with DCs stimu-lated in the presence of AEE and 2Ph. Human monocyte-derived DCs were stimulatedwith IL-1β (10 ng/ml), TNF (50 ng/ml) and LPS (500 ng/ml) in the presence of AEE at10 μg/ml (AEE-DC), 2Ph at 10 μg/ml (2Ph-DC) or 0.1% DMSO (DMSO-DC) for 2 daysand subsequently co-cultured with allogeneic CD4+ T cells for 6 days. Supernatantswere collected and the levels of IL-6 (a), IL-23 (b) and IL-12p40 (c) secretion weremeasured by ELISA with data given as the mean ± SEM. p-Values indicate statisticaldifference between DCs stimulated in the presence of AEE or 2Ph as compared withDCs stimulated in the presence of DMSO (one way ANOVA).

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DCs stimulated in the presence of AEE or 2Ph secreted reducedlevels of IL-12p40 and there was a tendency of reduced IL-12p40 se-cretion of AEE-DCs or 2Ph-DCs following co-culture with allogeneicCD4+ T cells. The allogeneic CD4+ T cells secreted lower levels ofIFN-γ following co-culture with the AEE-DCs and 2Ph-DCs. As IL-12is the main inducer of Th1 polarization with subsequent IFN-γ secre-tion by the T cells [27], the reduction in IFN-γ secretion by the CD4+ Tcells observed in this study is most likely resulting from a reducedability of the DCs stimulated in the presence of AEE or 2Ph to secreteIL-12.

As stimulation of the DCs in the presence of AEE or 2Ph had no ef-fect on IL-10 secretion, the reduced levels observed in the superna-tants of the co-cultured allogeneic CD4+ T cells and DCs were mostlikely the result of reduced secretion by T cells but not DCs.

DCs were stimulated in the presence of SbV and we found that itdid not affect the stimulation of DCs, or have any synergistic or antag-onistic effect when it was used in combination with AEE, as deter-mined by measuring expression of costimulatory molecules orcytokine secretion. This observation is also in line with previous find-ings and indicates that SbV has no effect on the immune response.

Following the co-culture with allogeneic CD4+ T cells, the DCsthat had been stimulated in the presence of AEE or 2Ph secreted re-duced levels of IL-6 and IL-23, both cytokines that affect Th17 polari-zation and hence IL-17 secretion of Th cells [28]. However, the levelsof IL-17 secretion by the allogeneic CD4+ T cells were not affected byco-culture with the DCs stimulated with AEE or 2Ph. Although secre-tion of IL-17 was not affected, other cytokines that are also induced byIL-6 and IL-23, such as IL-21 and IL-22, might be affected by the re-duced levels of IL-6 or IL-23 [28]. Th17 differentiation is also promot-ed by other cytokines which may be counteracting the effect of IL-6and IL-23, resulting in unchanged levels of IL-17.

DC maturation and activation can be inhibited by some classic im-munosuppressive drugs, including glucocorticoids, vitamin D3,rapamycin and cyclosporine A [29–32] as observed in this study bythe reduced IL-12p40 secretion of the DC following stimulation inthe presence of vitamin D3. In addition, several studies have reportedthat extracts from plants are able to affect DC stimulation [33–37a].Our results suggest that AEE and 2Ph interfere with LPS-induced stim-ulation of DCs, which in turn decrease the ability of DCs to stimulateallogeneic T cells towards the Th1 pathway. A number of signalingpathways are involved in LPS-induced DC activation and maturation,including MAP kinase, PI3 kinase, p38 SAP kinase, and NF-κB path-ways [38,39]. Whether NF-κB signaling pathway or other pathwaysare affected in this case remains to be determined and will be thenext part of this project.

In conclusion, our study provides new and valuable insight intothe action of AEE as immunomodulatory compound, besides itsleishmanicidal activity, indicating that treatment of DCs with AEEleads to reduced inflammatory cytokine production, and suppressesT cell priming capacity of the DCs. Understanding the immunomodu-latory effects of AEE will provide new and useful information whenconsidering it as an alternative treatment for leishmaniasis.

Disclosures

The authors have no financial conflict of interest.

Acknowledgments

The authors wish to acknowledge Dr. Alberto Giménez for provid-ing the extracts and to Consejo Indígena del Pueblo Tacana (CIPTA),Tumupasha and Rogelio Chuqui from the Tacana community ofSanta Rosa de Maravilla, for collection of plant material. We arethankful to Swechha Mainali Pokharel for the technical support. Thiswork was financially supported by the Swedish International Devel-opment Cooperation Agency (SIDA).

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