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Cellular immune polarization to a cell phenotype Th-1 and T-Reg is induced after the treatment of human dendritic cells infected by Leishmania with the compound 11α,19β-dihydroxy-7-acetoxy-7-deoxoichangin

Lucy G. Delgado1*, Diana S. Granados-Falla1, 2, Carlos Coy3 and Luis Cuca3
  • 1 Universidad Nacional de Colombia, School of Sciences, Colombia
  • 2 Universidad Nacional de Colombia, School of Sciences, Colombia
  • 3 Universidad Nacional de Colombia, School of Sciences, Colombia

Parasites of the genus Leishmania (Kinetoplastide order, family Trypanosomatidae) are responsible for clinical manifestations collectively known as leishmaniasis, a disease that is transmitted by the bite of hematophagous phlebotomine sand flies of the genus Lutzomyia and Phlebotomus (new and old world, respectively) which occurs in 98 countries endemically (located mainly in tropical and subtropical areas around the world), reporting about 2 million new cases of this disease (1, 2), which despite to the efforts and the progress made in recent years to control it, has shown an increasing trend. In this sense, the high toxicity, the prolonged therapeutic regimens as well their routes of administration and the emergence of resistant parasites to the drugs commonly used for the treatment (3-6), are reasons that shows that the therapy for controlling this disease is still far from be ideal, making clear the need to develop alternative therapies leading to a more secure and adequate control of this disease, for which the rational study of bioactive molecules with leishmanicidal activity and immunomodulatory (given the importance of the immune system in the natural control of leishmaniasis) (7-9) is an a approach suggested by the World Health Organization (WHO) available to address this problem (3, 10, 11).

Several studies for the evaluation of the immune response generated against natural infection and the "required" for the appropriate resolution of the infection caused by Leishmania parasites have been developed in order to design therapies that promote a favorable response and which generate memory cells. In this respect, it has been proposed that by using bioactive molecules with leishmanicidal and immunomodulatory activity can be achieve that "ideal" response, reason by which in the present study we evaluated the ability of 11α,19β-dihydroxy-7-acetoxy-7-deoxoichangin (a natural compound derived from the stem bark of Raputia heptaphylla, a species belonging to the family Rutaceae, whose genus has been traditionally used to treat the cutaneous presentation of the disease in some parts of South America) to activate dendritic cells (DCs) and to polarize the effector response (using for this purpose DCs infected in vitro with L. panamensis as well their T cells, for evaluate the expression of surface markers, the production of nitric oxide and cytokines involved in inflammatory processes by flow cytometry).

Briefly, heparinized peripheral blood mononuclear cells (PBMCs) obtained from healthy volunteers with medical history of the disease were centrifuged at 2,500 rpm for 15 minutes, and the leuko-platelet layer (buffy coat) was collected and diluted in RPMI-1640 (Gibco BRL-Life Technologies Inc.) for being used to prepare the density gradient by adding 1 volume of the ficoll-hypaque reagent (Invitrogen, USA) to 2 volumes of diluted blood sample, and the gradients were centrifuged for 30 minutes at 3,000 RPM at ambient temperature. Then, the PBMCs were cultured for 2 h at 37 °C in sterile Petri dishes (TPP, Techno Plastic Products, Switzerland) using RPMI 1640 medium (Gibco BRL-Life Technologies Inc., Grand Island, NY, USA) supplemented with 2 mM L-glutamine (Gibco BRL-Life Technologies Inc.), 1% non-essential amino acids, 1,000 U/ml penicillin, 0.1 mg/ml streptomycin, 0.25 ug/ml amphotericin B (Sigma Chemical Co., St. Louis, MO, USA), 24 mM sodium bicarbonate (Sigma Chemical Co.), 25 mM HEPES (Gibco BRL-Life Technologies Inc.) and 10% of AB negative human plasma, and time after which, the non-adherent fraction was collected and stored and the adherent fraction was cultured in the presence of human recombinant cytokines (GM-CSF and IL4 - BD Biosciences).

Aliquots of the obtained DCs were infected with promastigotes of Leishmania (Viannia) panamensis (strain MHOM/88CO/UA140, kindly donated by Dr. Sara Robledo from the Program for the Study and Control of Tropical Diseases; PECET - Programa de Estudio y Control de Enfermedades Tropicales, of the Universidad de Antioquia, Colombia) and once was established the infection, the cells were treated with several concentrations of the assessment compound (using for this purpose concentrations near to the EC50 previously found for this compound)

Then for the evaluation of the immunomodulatory activity was performed the determination of nitric oxide (by using DAF-FM diacetate solution - Invitrogen, USA), the quantification of IL-8, IL-1β, IL-6, IL-10, TNFa and IL-12p70 in supernatants cultures (using the kit of cytometric bead array – BD Biociencies) and the evaluation of the cell surface marker HLA-DR, CD209, CD83, CD3, CD4, CD8 and CD25 by Flow Cytometry (FacsCanto II - BD, Biosciences) following for all the cases the recommendations of supplier.

Here was observed that additional to the antileishmanial activity evidenced (only over amastigote form) this compound is also capable of induced a significant increase (when compared the response induced against untreated infected cells) in the production of pro-inflammatory soluble mediators, such as interleukin (IL) 12 (IL12p70) and Tumor Necrosis Factor alpha (TNF-α) (with p. value of 0.0480 and 0.012, respectively), along with the molecule of nitric oxide (p. value 0.0490), events accompanied by a significant increase in the expression on the number and amount of the human leukocyte antigen (HLA) class II-DR (HLA-DR) (p. value of 0.0141 and 0.0364, respectively) as also an increase (although not significant) on the expression of CD83 molecule (with a mean of 1993 + 994 MIF vs 558 + 324 in infected and treated DCs against infected without treatment DCs) (Figure 1 and 2)

Also, was observed a polarization of effector cell response towards a Th-1 and T-Reg phenotype (evidencing a tendency to increase the production levels of the cytokines IL-2 (mean of 9.86 vs 7.37 UI/mL for cells exposed to infected and treated DCs against infected cells without treatment), IFN-ɣ (mean 177.3 vs 170.3 UI/mL, respectively) and IL-10 (mean of 23.7 vs 22.8 UI/mL), with no found of detectable levels of TGF-β in the cells exposed to infected and treated DCs - contrary event to the evidence in untreated infected cells, where it was determined the presence of 232.1 pg/mL – (Figure 3), which according to reports from literature these phenotypes promotes the development of an adequate response against this intracellular pathogen.

These findings are consistent with previous evidence which show the compound's ability to induce the control of the Leishmania infection on in vitro and in vivo assays, showing an antileishmanial activity on antigen presenting cells (APCs) infected without any activity against extracellular form of the pathogen, as well as the resolution of skin lesions or total cure at least 50% of the animals tested (12-14).

We emphasize the potential use of the isolated compound 11α,19β-dihydroxy-7-acetoxy-7-deoxoichangin, a molecule that presents an antiparasitic and immunomodulatory activity capable to "reactivate" the infected phagocytic cells - in which their natural microbicides processes are "frozen" due to mechanisms of the pathogen - thereby promoting the development of an immune response aimed to the proper resolution and perhaps durable of cutaneous leishmaniasis.

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Figure 2
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Figure 3
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Acknowledgements

This work has been supported by the Administrative Department of Science, Technology and Innovation of Colombia - Colciencias (project “Immunomodulatory effect of plant extracts with potential leishmanicidal: confirmatory studies in vitro and preliminary in vivo”)

References

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13. Coy Barrera CA, Coy Barrera ED, Granados Falla DS, Delgado Murcia G, Cuca Suarez LE. seco-limonoids and quinoline alkaloids from Raputia heptaphylla and their antileishmanial activity. Chemical & Pharmaceutical Bulletin (2011) 59(7):855-9. Epub 2011/07/02. doi: JST.JSTAGE/cpb/59.855 [pii]. PubMed PMID: 21720036.
14. Granados-Falla D, Gómez-Galindo A, Daza A, Coy-Barrera C, Cuca L, Robledo S, et al. 11, 19-dihydroxy-7-acetoxy-7-deoxoichangin derived from Raputia heptaphylla, promotes the control of cutaneous leishmaniasis in hamsters (as experimental model in vivo). Submitted to Planta Medica (2013).

Keywords: Leishmaniasis, Dendritic Cells, Inmunomodulation, Treatment., seco-limonoid, natural compounds

Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013.

Presentation Type: Abstract

Topic: Host-pathogen interactions

Citation: Delgado LG, Granados-Falla DS, Coy C and Cuca L (2013). Cellular immune polarization to a cell phenotype Th-1 and T-Reg is induced after the treatment of human dendritic cells infected by Leishmania with the compound 11α,19β-dihydroxy-7-acetoxy-7-deoxoichangin. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.01024

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Received: 30 Jun 2013; Published Online: 22 Aug 2013.

* Correspondence: Dr. Lucy G Delgado, Universidad Nacional de Colombia, School of Sciences, Bogotá, Colombia, lgdelgadom@unal.edu.co