NATURAL PRODUCTS

DOI: 10.17533/udea.vitae.v22n1a02

MURINE INVARIANT NATURAL KILLER T CELLS RECOGNIZE GLYCOLIPIDS DERIVED FROM EXTRAC TS OF THE LICHEN Stereocaulon ramulosum

CÉLULAS T ASESINAS NATURALES INVARIANTES MURINAS RECONOCEN GLICOLIPIDOS DERIVADOS DE EXTRACTOS DEL LIQUEN Stereocaulon ramulosum

Andrés BAENA, M.Sc, Ph.D.^1,2,*, Lina GÓMEZ-GIRALDO, B.Sc.,^1,3 Wilton A. GÓMEZ, B.Sc.^1,3 Carlos A. PELÁEZ, M.Sc, Ph.D.⁴

¹ Grupo de Inmunología Celular e Inmunogenética GICIG, Departamento de Microbiología y parasitología, Facultad de Medicina, Universidad de Antioquia UdeA, Calle 70 No.52-21, Medellín, Colombia

² Sede de Investigación Universitaria, Facultad de Medicina, Universidad de Antioquia, Cra. 53 No. 61-30, Lab 510, Medellín-Colombia

³ L.G.G and W.G.G contributed equally to the development of this work

⁴ Grupo Interdisciplinario de estudios moleculares GIEM, Departamento de Microbiología y parasitología, Facultad de Medicina, Universidad de Antioquia UdeA, Calle 70 No.52-21, Medellín, Colombia

^* Corresponding author: andres.baenag@udea.edu.co.

Recibido: Febrero 06 de 2015

Aceptado: Mayo 05 de 2015

ABSTRACT

Background: Invariant natural killer T cells (iNKT ) can be activated by certain types of glycolipids that have the potential to generate adjuvant effects which could be used to develop effective and safe immunotherapies. Many of these glycolipids have been isolated from natural organisms, but there is a great amount of these organisms completely unexplored as a source of these types of compounds. Some of these organisms are lichens which are complex symbiotic organisms that have been showed to contain glycolipids. Objectives: We decide to test if glycolipids isolated from lichens would be able to activate iNKT cells in vitro and in vivo. Methods: We have used extracted glycolipids from 43 different species of lichens from Colombia. We have used iNKT hybridoma cells, C57BL/6 mice, IL-2 ELISA and the B16 melanoma to test for the adjuvant capabilities of glycolipids isolated from lichens. Results: In this study we have found two glycolipids with the capacity to activate iNKT cells in vitro. One of the glycolipids was able to activate iNKT cells in vivo, and was competent to induce protection against the B16 melanoma in the mouse model. Conclusions: We propose a possible chemical structure for a novel glycolipid called β-GalCer-lich (1) derived from the lichen Stereocaulon ramulosum.

Keywords: iNKT s; Glycolipids; CD1d; Lichens; Adjuvant.

RESUMEN

Antecedentes: Las células asesinas naturales T (iNKT ) pueden ser activadas por ciertos tipos de glicolípidos que tienen el potencial para generar efectos adyuvantes los cuales pueden ser usados para desarrollar inmunoterapias efectivas. Muchos de estos glicolípidos han sido aislados de organismos naturales, pero hay una gran cantidad de organismos completamente inexplorados como fuente de este tipo de compuestos. Algunos de estos organismos son los líquenes, los cuales son organismos simbiontes complejos para los que se ha mostrado que contienen glicolípidos. Objetivos: Nosotros decidimos probar. si los glicolípidos aislados de líquenes podrían ser capaces de activar alas celulas iNKT in vitro e in vivo. Métodos: Nosotros hemos extraído glicolípidos de 43 especies de líquenes de Colombia. Nosotros hemos usado células de un hibridoma de iNKT s, ratones C57BL/6, ELISA para IL-2 y el melanoma B16 para probar la capacidad adyuvante de los glicolipidos aislados de los líquenes. Resultados: En este estudio nosotros hemos encontrado dos glicolípidos con la capacidad de activar iNKT s in vitro. Uno de los glicolípidos fue capaz de activar células iNKT in vivo, y fue competente para inducir protección contra el melanoma B16 en el modelo de ratón. Conclusiones: Nosotros proponemos una posible estructura química para el nuevo glicolípido llamado β-GalCer-lich (1) derivado del liquen Stereocaulon ramulosum.

Palabras clave: iNKT s; Glicolípidos; CD1d, Líquenes; Adyuvante.

INTRODUCTION

It is very interesting that αGalCer, which is the most potent compound known to activate iNKT cells (14), was isolated presumably from a cyanobacteria algae (present in a marine sponge) that is closely related to the algae present in lichens (4, 15, 16). Lichens are symbiotic organisms consisting of a fungus and cyanobacteria algae that have been used in traditional medicine for the treatment of diseases such as tuberculosis (17, 18). Moreover, there are different reports in which lichen extracts have been showed to have anticancer and antibiotic properties (19-21).

The fact that Colombia is one of the countries with the highest diversities of lichens in the world, with approximately 1500 species, allow us to obtain good number of lichen samples (22, 23). In this study we have isolated glycolipids from 43 different species of lichens and found 2 of them with the capacity to activate iNKT cells in vitro. As we shown in this paper, the glycolipid extracted from Stereocaulon ramulosum (S.ramulosum) was able to activate iNKT cells in vivo and confered protection as an adjuvant in the mouse model against the B16 melanoma. Finally, we propose a possible chemical structure for the glycolipid isolated from S. ramulosum that could be produced synthetically in the near future as a potential vaccine adjuvant.

METHODS

Mice

All mice used in the study were purchased from Jackson Laboratories (Bar Harbor, ME). C57BL/6 female mice were maintained under specific pathogen-free conditions and were used for experiments at 6-8 weeks of age. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of the Universidad de Antioquia.

Cell culture

For in vitro cultured, murine cells were maintained in complete RP MI-1640 medium (Invitrogen, Carlsbad), supplemented with 10 mM HEPES pH 7.5, 1% L-glutamine, 1% nonessential amino acids, 1% Sodium Pyruvate 55 μM 2-mercaptoethanol, 20 μg/ml gentamicin (Gibco), 10% heat-inactivated fetal calf serum (FCS) and beta-mercapto-ethanol. The cell lines were maintained in an incubator with a humidified atmosphere containing 5 % CO2 at 37°C.

Bone Marrow-Derived Dendritic Cells(BMDCs)

iNKT cell hybridoma Stimulation

For dose titration experiments, BMDCs and DN3A4-1.2 NKT hybridomas (M. Kronenberg at La Jolla Institute for Allergy and Immunology and S.A. Porcelli at the Albert Einstein College of Medicine) were cultured at a 2:1 hybridoma:BMDC ratio (murine iNKT hybridoma cells at 5x10⁴ cells/well in 96-well plates were stimulated with the 1x10⁵ cells/well pulsed BMDCs with the indicated doses of glycolipids) in the presence of 0.5 mg/ml LPS (Escherichia coli O26:B6; Sigma-Aldrich). Supernatants were harvested after 16 h at 37°C, and levels of murine IL-2 secretion were determined in the supernatants by ELISA. To block antigen presentation we use the anti-CD1d 1B1 (BD Biosciences) antibody that was added to the BMDCs at 10 ug/ml 30 min before addition of the iNKT cells and after 24hrs the supernatant was tested for IL-2 production by ELISA.

Reagents and glycolipids

Glycolipids were prepared as solutions for injection in vehicle (PBS with 0.1 % DMSO and 0.05 % of Tween-20). For in vitro assays, glycolipids were dissolved in 10 % DMSO at 500 mM, sonicated in a water bath sonicator for 5 min and then diluted directly into prewarmed (37°C) culture medium at a final concentration of 20 ng/ul. Glycolipid MGDG and αGalCer were purchased from Avanti Polar Lipids and Funakoshi co., respectively. The αGalCer was used as a positive control at a final concentration of 100 ng/ml and DMSO was used as a negative control for all the in vitro experiments.

Lichens collection

The lichens were collected in the following locations: Barbosa, Antioquia-Colombia (N 06° 27.421'W 0.75° 18.22'; 1300 mts), Santa Elena, Antioquia- Colombia (N 06° 15.821´W 0.75° 29.465´; 2325 mts), Envigado, Antioquia-Colombia (N 06° 16.061´ W 0.75° 29.682´; 2400 mts) and Belmira, Antioquia-Colombia (N06°31.957´ O 075° 38.662´; 2800 mts). We collected approximately 20 gr of 43 different species of lichens, for which a representative piece of each sample was dried and stored in a small collection, which was used to stablish the lichens classifications. Each sample was accompanied by a set of photographs as evidence of the place where the sample was found and also to check for taxonomic details of the fresh sample; the rest of the material was used for the glycolipid extraction.

Glycolipid extraction

The lichens were washed multiple times with water and purified from contaminants using tweezers and then they were left at room temperature for 24 hrs. After this step, the lichens were completely dried in an oven at 105 °C overnight. The samples were blended to get a particulate material to optimize the extraction. All the glycolipids were extracted with CH₂Cl₂:MeOH : ddH₂O (1:2:1) in a shaker for 24 hrs as a modification of the Bligh and Dyer protocol (25). The liquid phase was separated from the particulate material. The samples were reextracted with CH2Cl2:MeOH : ddH2O (1:1:1) for 7 hrs and then the phases were allowed to separate for 18 hrs before the lower dichloromethane was removed and filtered through Whatman filter paper and dried with nitrogen gas. Finally, the samples were store in sterile glass containers at room temperature and in the dark.

For the soxhlet extraction the material was refluxed over the thimble in a standard soxhlet extraction apparatus for 3 hrs using Hexane, chloroform and methanol. The glycolipid containing solvent was then recovered in the attached round-bottom flask which was used to dry the lipid under rotary vacuum evaporation (<37 °C). The glycolipid residue was re-dissolved, transferred to a sterile small glass container, and then solved evaporated.

Supernatant levels of IL-2 were measured by ELISA using capture and biotinylated detection antibody pairs and streptavidin-horseradish peroxidase (BD PharMingen) with substrate reagent pack (R&D). IL-2 standard was obtained from Pepro- Tech (Rocky Hill, NJ). 96 well plates were coated overnight at 4 °C with the capture antibody and blocked for nonspecific binding sites by adding 1 % bovine serum albumin in phosphate-buffered saline (PBS) for 1 hr. The supernatants were added for 2 hrs and then the detection biotnylated antibody was added for 1hr. After these the streptavidine-HRP was added for 45 min. Finally, the substrate was added and the reaction was stopped with sulfuric acid. All the washes were done with PBS 1X and 0.05 % of Tween 20. Optical density at 450 nm was measured 5 minutes after stopping the reaction. The IL-2 concentrations were calculated based on the standard curve obtained in each assay (Supl. Fig. 2A, 2B, 2C and 2D) (All Supplementary material are presented at the end of the paper).

TLC (Thin layer chromatography)

The crude extract was dissolved in a minimum amount of CH₂Cl₃:MeOH (2:1) and , applied to a 5 mm preparative TLC silica plates (Alltech Associates) and eluted with CHCl3:MeOH :AcOH :H2O (100:20:12:5). The plate was divided into three section based on polarity (top, S5 middle, and bottom). Each section was scraped off the plate and extracted with CHCl3:MeOH (5:1). The resulting solution was concentrated and further purified by preparative TLC (0.5 mm): the top section was eluted in CH2Cl3:MeOH (98:2). After running solvent front to within 1 cm of the top, the plates were dried and analyzed by orcinol staining. The orcinol was prepared taking 100 mg of orcinol monohydrate (Sigma-Aldrich) mixed with 12,5 ml of MEOH , 2,5 ml of ddH₂O and 5 ml of H₂SO₄ (98%). The orcinol was sprayed over the dried plates and heated to 120 °C for 5 min. After this procedure the glycolipids can be easily distinguishable as yellow spots. The spots attached to the silica were scraped from the plate and re-extracted with CH₂Cl₃:MeOH (1:1) and dried with nitrogen gas. The maximum amount of material that we got per individual plant was 10 grams. After the extraction with chloroform /methanol we got between 42 mg to 200 mg. From this material, we purified the glycolipids by using TLC-Silica from where we obtain from 20 to 250 μg of final material.

NMR

The samples were re-suspended in 1ml of CD₃OD (deuterated MeOH , NMR grade). For the analysis we used an NMR BRUKER 300MHz for 12 hrs for 1H-NMR at University of Antioquia.

Mass-Spectrometry

The Mass-Spectrometry analyses were done at the University of Kansas (Mass spectrometry and analytical proteomics laboratory,1251 Wescoe Hall Lawrence, KS). Direct Infusion of glycolipid samples and acquired on a Qtof type instrument Waters Synapt G2. The samples were re-suspended in ammonium acetate (NH₄OAc) or Chloroform and methanol (2:1). We use direct infusion in negative mode with high energy fragmentation and α-GalCer (Cayman) and Monogalactosyl-Diacylglycerol (MGDG, Matreya) were used as controls for each sample analyzed (Supl. Fig. 4A and B).

B16 melanoma

To test for the activity of the lichen glycolipids we used the tumor suppression by iNKT cells in a B16 melanoma lung metastasis model. The B16 cells were grown in complete media until they reach a confluence of 70 %. B16 melanoma cells (5x10⁵ cells /mouse) were injected into a tail vein of C57BL/6 mice, and twelve days after, the mice were sacrifice to measure the pulmonary metastasis as number of nodules per lung. Mice injected (i.p.) with the glycolipid solutions each received 0.2 ml (containing 4 μg of each glycolipid) for a glycolipid dose that was previously heated at 80 °C for 5 minutes four times, every two days after receiving the B16 melanoma.

Antibodies to CD1d-APC, MHC class II (IA/ I-E)-FITC, CD11c-PE, CD3, Vα14-FITC and Vβ8.2-PE were purchased from BD Biosciences as fluorescent dye conjugates. Samples were analyzed using the FACS-Calibur instrument at the Albert Einstein College of Medicine, (BD Biosciences). The data was analyzed with Flowing 2.5.1 and Flowjo 7.6.

Statistical Analysis

Two way ANOV A with Bonferroni´s multiple comparisons was used for comparing groups of three or more glycolipid treatments was used to generate p values for selected pairwise comparisons, and p < 0.05 were considered significant. GraphPad Prism 5.0 software was used for statistical analyses. All experiments were performed in triplicate, and the results are presented as mean values with error bars representing the standard deviation (S.D.).

RESULTS

Evaluation of the iNKT cell activation by glycolipids derived from lichens

We have isolated glycolipids from a collection of different species of lichens from Colombia using a modification of the Bligh and Drier protocol and TLC (25). After the orcinol staining of the TLC plates, we were able to detect and isolate 41 glycolipids (Fig. 1A) (See methods section). To gain insight into the iNKT cells potential activity of these lichen glycolipids, we used an assay system to screen for lipid activity based on a single-TCR-specific iNKT cell hybridoma (DN3A4 1.2) that was cultured together with bone marrow derived dendritic cells BMDCs (Supl. Fig. 1A, 1B). For the selection of glycolipids, we assumed positive activity for glycolipid extracts with five standard deviations above the basal activity of the negative control. Among the panel of lichen glycolipids extracts tested, only glycolipid 3 and glycolipid 26 were differentially stimulatory (Fig. 1B, 1B, Supl. Fig. 3).

Glycolipid 3 is derived from de lichen Stereocaulon ramulosum

Since glycolipid 3 showed the highest activity and reproducibility, we decided to focus on this glycolipid for the rest of the paper. To see if we can obtain more glycolipid 3 from the collected material, we performed the isolation using the traditional soxhlet technique, with tree different solvents (chloroform-42, Hexane-43 and methanol- 44) (Fig. 2A). We found activity with the crude extracts for chloroform and methanol (Fig. 2A). After seeing this result we decided to do a TLC with the chloroform-42 and we were able to isolate four spots (54,55,56 and 57) from which 57 gave us the highest activity (Fig. 2B), and correspond to the same spot as for glycolipid 3 with an Rf of 0,9 (data not showed). We were able to get more material from the lichens by using the soxhlet method but in terms of iNKT cell activity for glycolipid 3 there were not significant differences between the two methods that were evaluated in the paper.

The activity of glycolipid 3 is CD1d dependent

Due to the fact that iNKT cells can be activated directly by the CD1d or indirectly by cytokines, we try to determine whether the activation of the glycolipid 3 was dependent on CD1d. Remarkably, the activity of glycolipid 3 was significantly reduced when a blocking antibody (anti-CD1d) was present, suggesting a major dependence on CD1d for this glycolipid during the activation of iNKT cells ((Fig. 2C). Notably, blocking the activity of CD1d for αGalCer cause a greater reduction of the production of IL-2 by the iNKT cells as compared with glycolipid 3 which could be explained by the concentration and purity of the αGalCer as compared to glycolipid 3 (Fig. 2C).

Glycolipid 3 has anti-tumor activity in vivo

Different glycolipids have been shown to promote antitumor immune responses in several cancer models trough the activation of iNKT cells (26-28). In order to determine if glycolipid 3 has in vivo activity, 18 Vitae a. Baena et al. we evaluate the effect of this glycolipid to control the growth of the mouse B16 melanoma. We performed an administration of αGalCer and glycolipid 3 in four doses every two days after the administration of the of B16 melanoma cells in mice. B16 melanoma cells (5x10⁵ cells /mouse) were injected into a tail vein of C57BL/6 mice, and twelve days later, the mice were sacrifice to measure the pulmonary metastasis represented by the number of nodules per lung (Fig. 3A). As shown in Fig. 3B, αGalCer and glycolipid 3 alone, both caused a very significant decreased in the number of pulmonary metastatic foci, although the reduction of glycolipid 3 was not as significant as for αGalCer. It is possible that the administration of a-GalCer results in a more rapid and massive activation of iNKT cells and a production of typical Th1 and Th2 cytokines as compared to glycolipid 3. The other possibility is that the concentration of αGalCer could be higher as compared to the concentration of the glycolipid 3. These results could be confirmed in the future with a synthetic version of glycolipid 3.

Glycolipid 3 is a new antigen derived from S.ramulosum now called β-GalCer-lich

After having reproducible results that showed activity in vitro and in vivo for glycolipid 3, we did an approximation to solve its chemical structure. We subjected the glycolipid extracts to high-resolution MS where we observed one main peak at 811.4 (Fig. 4A). In order to calculate the correct molecular weight based on the MS we have to subtract an NH₄ (-18) and a hydrogen atom (-1) due to the negative mode which gave us a final molecular weight of 792.4 which is consistent with the size range of multiple reported ceramides. A set of 1D ¹H-NMR experiments for glycolipid 3 yielded resonances consistent with other glycolipids previously reported. The ¹H-NMR spectrum shows a peak R1 of 7,309 that correspond to the NH- group characteristic of a ceramide (Fig. 4B). Moreover, the spectrum shows an anomeric proton (R2 peak) with a chemical shift of 5.3 consistent with a β-linkage of the sugar to the ceramide; the group of peaks called R3 represents the sugar moiety associated with the ceramide; the groups of peaks denoted as R4 correspond to different carbons from distinct parts of the chemical structure. Finally, the group of peaks R5 represents the tails of carbons that form the ceramide (Fig. 4B). Many of the structure assignments were based on the foot-printing technique and by comparisons with the ¹H- NMRs of the closest compound αGalCerBf (29).

We use the predictor algorithm of the software MestReNova (version 9.0.1, 2014, MestReNova Research S.L.) to obtain the possible chemical structure for glycolipid 3 applying the molecular weight and the functional groups to match the ¹H-NMR spectrum. Our data rely on the proton chemical shift prediction which can have great differences as compared to the ¹H-NMR of the natural compound due to the unpredictable dependency on average solution conformational effects, although we found very close similarities. The predicted ¹HNMR spectrum shows chemical shifts (δ): 7.309, 5.346, 3.963, 3.712, 3.536, 2.665, 1.649 and 1.299 which are shared between the two spectrums (Supl. Fig. 5A). We observed more dissimilar patterns at the level of the fatty acid tails and the sugar which could be affected by some contaminants present after our glycolipid purification method.

The closest predicted ¹H-NMR spectrum to the one obtained for glycolipid 3, gave us a chemical structure of a β-galactosyl-ceramide with a possible C16:0 fatty acid and a C24 sphingosine that we called β-GalCer-lich (1) (Fig. 4D). This compound has a spectrum that almost completely overlaps the NMR spectrum that was showed for glycolipid 3 (Fig. 4C).

This is in accordance with a previous report of a β-D-galactopyranosyl ceramide that was isolated from the lichen Ramalina celastri (11) which may originate from the algae. The most interesting feature of the glycolipid from R. celastri is that the most abundant fatty acids in this lichen are C16:0 with 36% and some of them with a molecular weight of 780 which is closely related to our β-GalCer-lich (1).

DISCUSSION

Here we identify a lichen derived glycosphingolipid antigen, β-GalCer-lich (1) that directly activates iNKT cells in a CD1d-restricted manner. To our knowledge, this is the first report of a lichen derived glycolipid antigen that directly activates iNKT cells, and therefore this study unveiled a new source of iNKT cell ligands. It is well known that iNKT TCRs can recognize β-linked glycolipid antigens via induced-fit molecular mimicry, in which the β-linked sugars are molded into an α-like conformation by the iNKT TCR (30-32). There are reports for β-linked glycolipids such us the glycolipid β-galactosylceramide (β-GalCer), which is structurally very similar to β-GlcCer that is also recognized by mouse and human NKT cells, albeit with apparently lower potency than the recognition of β-GlcCer. Our β-GalCer-lich (1) (C16:0) is closely related to the β-GalCer (C18:0) which also exert tumor-specific cytotoxicity in vitro but has two more carbons (33, 34). It was indicated that the growth of transplanted tumors is suppressed in mice with β-ceramides with reduced fatty acid length dawn to C12, but not in those treated with β-ceramides with less carbons such us C8 (34). Another report showed results with β-GalCer C12 and β-GalCer C26 that can effectively activate iNKT cells at high concentrations in vitro and vivo similar to the prototypical αGalCer (35).

Two signals have an important role in iNKT cell activation during microbial infection: the first is a lipid antigen presented by CD1d to the iNKT cell TCR, and the second is an inflammatory cytokine, such as IL-12 (4). We did not evaluate the second alternative directly which could be present during the activation effect of our β-GalCer-lich (1) compound in vivo. Moreover, it has been showed before that αGalCer has an adjuvant effect by the induction of dendritic cell maturation, which is not seeing for other type of compounds such us αGalCerBf. To observe a good activity for our β-GalCer-lich (1) we have to use LPS in our in vitro experiments to mature the BMDCs if we want to get maximum activity in spite of knowing that the DN3A4 hybridoma does not respond to IL-12 derived from the treatment (36).

It would be interesting to compare if the β-GalCer-lich (1) is more potent that the β-GlcCer and the β-GalCer in vitro and in vivo, as the length and degree of saturation of the fatty acid tails has a considerable effect on the activity of a glycolipid antigen. This glycolipid comparison might show differences in antigen processing, glycolipid loading and TCR binding affinity. We are aware that this β-GalCer-lich (1) could be just an approximation to the real compound present in the lichen, and that the only way to be certain about the chemical structure is to synthetize the compound and be able to reproduce the activity as it was done for other glycolipids (37, 38).

We acknowledge funding from: "Programa de Sostenibilidad 2014-2015, Universidad de Antioquia" and CODI-"Programa primer Proyecto profesor vinculado-Universidad de Antioquia". We appreciate the support by all GICIG members especially with Luis F. Barrera for the advice given during the development of this project. We are in debt with Robinson Ramirez, Paula Correa and to all the other members of the GIM group for all his support with animal handling. We are grateful to Steven A. Porcelli and Leandro Carreño for the iNKT cell hybridoma. We want to thank COR ANTIOQ UIA for the authorization to obtain samples for this study. There is no "Conflict of interests" of authors, with the research results..

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