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Functional Validation of ABCA3 as a Miltefosine Transporter in Human Macrophages

IMPACT ON INTRACELLULAR SURVIVAL OF LEISHMANIA (VIANNIA) PANAMENSIS*
  • Luuk C.T. Dohmen
    Affiliations
    From the Centro Internacional de Entrenamiento e Investigaciones Médicas, Cra. 125 # 19–225 Cali, Colombia

    Division of Pharmacoepidemiology and Clinical Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3508 TB Utrecht, The Netherlands
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  • Adriana Navas
    Affiliations
    From the Centro Internacional de Entrenamiento e Investigaciones Médicas, Cra. 125 # 19–225 Cali, Colombia
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  • Deninson Alejandro Vargas
    Affiliations
    From the Centro Internacional de Entrenamiento e Investigaciones Médicas, Cra. 125 # 19–225 Cali, Colombia
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  • David J. Gregory
    Affiliations
    Molecular and Integrative Physiological Sciences, Harvard T. H. Chan School of Public Health, Boston, Massachusetts 02115
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  • Anke Kip
    Affiliations
    Division of Pharmacoepidemiology and Clinical Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3508 TB Utrecht, The Netherlands

    Department of Pharmacy and Pharmacology, Antoni van Leeuwenhoek Hospital/Slotervaart Hospital, 1066 CX Amsterdam, The Netherlands
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  • Thomas P.C. Dorlo
    Affiliations
    Division of Pharmacoepidemiology and Clinical Pharmacology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, 3508 TB Utrecht, The Netherlands

    Department of Pharmaceutical Biosciences, Uppsala University, 751 24 Uppsala, Sweden
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  • Maria Adelaida Gomez
    Correspondence
    To whom correspondence should be addressed. Tel.: 57-2-5552164; Fax: 57-2-5552638
    Affiliations
    From the Centro Internacional de Entrenamiento e Investigaciones Médicas, Cra. 125 # 19–225 Cali, Colombia
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  • Author Footnotes
    * This work was supported, in part, by COLCIENCIAS Grants 250-2010, Code 2229-519-28930, and 738-2009, Code 2229-49326161, the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases Re-entry Grant B00032, and NIAID, National Institutes of Health Grant R01AI104823. The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Open AccessPublished:February 22, 2016DOI:https://doi.org/10.1074/jbc.M115.688168
      Within its mammalian host, Leishmania resides and replicates as an intracellular parasite. The direct activity of antileishmanials must therefore depend on intracellular drug transport, metabolism, and accumulation within the host cell. In this study, we explored the role of human macrophage transporters in the intracellular accumulation and antileishmanial activity of miltefosine (MLF), the only oral drug available for the treatment of visceral and cutaneous leishmaniasis (CL). Membrane transporter gene expression in primary human macrophages infected in vitro with Leishmania Viannia panamensis and exposed to MLF showed modulation of ABC and solute liquid carrier transporters gene transcripts. Among these, ABCA3, a lipid transporter, was significantly induced after exposure to MLF, and this induction was confirmed in primary macrophages from CL patients. Functional validation of MLF as a substrate for ABCA3 was performed by shRNA gene knockdown (KD) in THP-1 monocytes. Intracellular accumulation of radiolabeled MLF was significantly higher in ABCA3KD macrophages. ABCA3KD resulted in increased cytotoxicity induced by MLF exposure. ABCA3 gene expression inversely correlated with intracellular MLF content in primary macrophages from CL patients. ABCA3KD reduced parasite survival during macrophage infection with an L. V. panamensis strain exhibiting low in vitro susceptibility to MLF. Confocal microscopy showed ABCA3 to be located in the cell membrane of resting macrophages and in intracellular compartments in L. V. panamensis-infected cells. These results provide evidence of ABCA3 as an MLF efflux transporter in human macrophages and support its role in the direct antileishmanial effect of this alkylphosphocholine drug.

      Introduction

      Leishmania parasites are the causative agent of cutaneous leishmaniasis (CL)
      The abbreviations used are: CL
      cutaneous leishmaniasis
      MLF
      miltefosine
      KD
      knockdown
      ABC
      ATP binding cassette
      PMA
      phorbol 12-myristate 13-acetate
      PBMC
      peripheral blood mononuclear cell.
      and visceral leishmaniasis, neglected tropical diseases with a combined annual incidence of an estimated 1.5 million cases globally (
      • Alvar J.
      • Vélez I.D.
      • Bern C.
      • Herrero M.
      • Desjeux P.
      • Cano J.
      • Jannin J.
      • den Boer M.
      • WHO Leishmaniasis Control Team
      Leishmaniasis worldwide and global estimates of its incidence.
      ). Among the scarce chemotherapeutic options available, antimonial drugs remain the first line treatment in most affected countries. However, loss of susceptibility against pentavalent antimonials shown in Leishmania isolates from both visceral leishmaniasis and CL patients, increased rates of therapeutic failure, and high toxicity, threaten its usefulness (
      • Vanaerschot M.
      • Dumetz F.
      • Roy S.
      • Ponte-Sucre A.
      • Arevalo J.
      • Dujardin J.C.
      Treatment failure in leishmaniasis: drug-resistance or another (epi-) phenotype?.
      ). An alternative treatment option is the alkylphosphocholine miltefosine (MLF), the only oral drug available and registered for the treatment of CL and visceral leishmaniasis (
      • Dorlo T.P.
      • Balasegaram M.
      • Beijnen J.H.
      • de Vries P.J.
      Miltefosine: a review of its pharmacology and therapeutic efficacy in the treatment of leishmaniasis.
      ). The response to MLF treatment during L. Viannia infections apparently varies among patients infected with different L. Viannia species, with cure rates that range from 60% in regions of Guatemala where L. V. braziliensis and Leishmania mexicana are common (
      • Soto J.
      • Arana B.A.
      • Toledo J.
      • Rizzo N.
      • Vega J.C.
      • Diaz A.
      • Luz M.
      • Gutierrez P.
      • Arboleda M.
      • Berman J.D.
      • Junge K.
      • Engel J.
      • Sindermann H.
      Miltefosine for new world cutaneous leishmaniasis.
      ), to 90% in Bolivia and Colombia in infections caused by L. V. braziliensis and L. V. panamensis, respectively (
      • Soto J.
      • Rea J.
      • Balderrama M.
      • Toledo J.
      • Soto P.
      • Valda L.
      • Berman J.D.
      Efficacy of miltefosine for Bolivian cutaneous leishmaniasis.
      ,
      • Rubiano L.C.
      • Miranda M.C.
      • Muvdi Arenas S.
      • Montero L.M.
      • Rodríguez-Barraquer I.
      • Garcerant D.
      • Prager M.
      • Osorio L.
      • Rojas M.X.
      • Pérez M.
      • Nicholls R.S.
      • Gore Saravia N.
      Noninferiority of miltefosine versus meglumine antimoniate for cutaneous leishmaniasis in children.
      ). Although it has been suggested that the therapeutic response to MLF is potentially linked to species-specific loss of susceptibility (
      • Escobar P.
      • Matu S.
      • Marques C.
      • Croft S.L.
      Sensitivities of Leishmania species to hexadecylphosphocholine (miltefosine), ET-18-OCH(3) (edelfosine) and amphotericin B.
      ,
      • Yardley V.
      • Croft S.L.
      • De Doncker S.
      • Dujardin J.C.
      • Koirala S.
      • Rijal S.
      • Miranda C.
      • Llanos-Cuentas A.
      • Chappuis F.
      The sensitivity of clinical isolates of Leishmania from Peru and Nepal to miltefosine.
      ), recent studies show a wide range of susceptibilities among clinical isolates of the same Leishmania species (
      • Fernández O.L.
      • Diaz-Toro Y.
      • Ovalle C.
      • Valderrama L.
      • Muvdi S.
      • Rodríguez I.
      • Gomez M.A.
      • Saravia N.G.
      Miltefosine and antimonial drug susceptibility of Leishmania Viannia species and populations in regions of high transmission in Colombia.
      ), and therapeutic failure has been reported in visceral leishmaniasis patients infected with drug-susceptible strains (
      • Rijal S.
      • Ostyn B.
      • Uranw S.
      • Rai K.
      • Bhattarai N.R.
      • Dorlo T.P.
      • Beijnen J.H.
      • Vanaerschot M.
      • Decuypere S.
      • Dhakal S.S.
      • Das M.L.
      • Karki P.
      • Singh R.
      • Boelaert M.
      • Dujardin J.C.
      Increasing failure of miltefosine in the treatment of Kala-azar in Nepal and the potential role of parasite drug resistance, reinfection, or noncompliance.
      ). We have recently shown that underexposure was associated with MLF treatment failure (
      • Dorlo T.P.
      • Rijal S.
      • Ostyn B.
      • de Vries P.J.
      • Singh R.
      • Bhattarai N.
      • Uranw S.
      • Dujardin J.C.
      • Boelaert M.
      • Beijnen J.H.
      • Huitema A.D.
      Failure of miltefosine in visceral leishmaniasis is associated with low drug exposure.
      ). Together, these findings indicate that parasite factors, other than classical drug resistance, and host factors are critical to the efficacy of MLF treatment.
      During human infection, Leishmania preferentially resides and replicates within macrophages. Thus, the direct activity of antileishmanial drugs is dependent upon effective drug delivery to host cells and to the intracellular compartments (phagolysosomes) inhabited by the parasite. However, which host factors contribute to the therapeutic response and how remain poorly understood. Our group and others have shown that transport and accumulation of drugs within human macrophages affect the antileishmanial activity of first line antimonials (
      • Gómez M.A.
      • Navas A.
      • Márquez R.
      • Rojas L.J.
      • Vargas D.A.
      • Blanco V.M.
      • Koren R.
      • Zilberstein D.
      • Saravia N.G.
      Leishmania panamensis infection and antimonial drugs modulate expression of macrophage drug transporters and metabolizing enzymes: impact on intracellular parasite survival.
      ,
      • Mookerjee Basu J.
      • Mookerjee A.
      • Banerjee R.
      • Saha M.
      • Singh S.
      • Naskar K.
      • Tripathy G.
      • Sinha P.K.
      • Pandey K.
      • Sundar S.
      • Bimal S.
      • Das P.K.
      • Choudhuri S.K.
      • Roy S.
      Inhibition of ABC transporters abolishes antimony resistance in Leishmania infection.
      ). The mechanisms regulating MLF trafficking within host macrophages and its role in drug exposure and direct drug activity against intracellular Leishmania remain unknown.
      We have recently observed that intracellular MLF concentrations in peripheral blood mononuclear cells isolated from miltefosine-treated CL patients followed a similar kinetic profile as plasma MLF concentrations (
      • Kip A.E.
      • Rosing H.
      • Hillebrand M.J.
      • Castro M.M.
      • Gomez M.A.
      • Schellens J.H.
      • Beijnen J.H.
      • Dorlo T.P.
      Quantification of miltefosine in peripheral blood mononuclear cells by high-performance liquid chromatography-tandem mass spectrometry.
      ). A trend of higher end of treatment intracellular concentrations versus plasma concentrations was observed, suggesting intracellular MLF accumulation in human leukocytes (
      • Kip A.E.
      • Rosing H.
      • Hillebrand M.J.
      • Castro M.M.
      • Gomez M.A.
      • Schellens J.H.
      • Beijnen J.H.
      • Dorlo T.P.
      Quantification of miltefosine in peripheral blood mononuclear cells by high-performance liquid chromatography-tandem mass spectrometry.
      ). Because MLF is an amphipathic phospholipid molecule, it is expected to cross passively over lipid membranes of the cell (
      • Ménez C.
      • Buyse M.
      • Farinotti R.
      • Barratt G.
      Inward translocation of the phospholipid analogue miltefosine across Caco-2 cell membranes exhibits characteristics of a carrier-mediated process.
      ,
      • Ménez C.
      • Buyse M.
      • Dugave C.
      • Farinotti R.
      • Barratt G.
      Intestinal absorption of miltefosine: contribution of passive paracellular transport.
      ). However, an additional carrier-mediated transport mechanism dependent on temperature and ATP has also been shown to contribute to MLF accumulation in human cells (
      • Ménez C.
      • Buyse M.
      • Farinotti R.
      • Barratt G.
      Inward translocation of the phospholipid analogue miltefosine across Caco-2 cell membranes exhibits characteristics of a carrier-mediated process.
      ). Overexpression of the ATP binding cassette (ABC) transporter MDR1 in cancer cell lines results in reduced sensitivity to MLF (
      • Rybczynska M.
      • Liu R.
      • Lu P.
      • Sharom F.J.
      • Steinfels E.
      • Di Pietro A.
      • Spitaler M.
      • Grunicke H.
      • Hofmann J.
      MDR1 causes resistance to the antitumour drug miltefosine.
      ). ABC transporters LMDR1/LABCB4 (
      • Pérez-Victoria J.M.
      • Pérez-Victoria F.J.
      • Parodi-Talice A.
      • Jiménez I.A.
      • Ravelo A.G.
      • Castanys S.
      • Gamarro F.
      Alkyl-lysophospholipid resistance in multidrug-resistant Leishmania tropica and chemosensitization by a novel P-glycoprotein-like transporter modulator.
      ,
      • Pérez-Victoria J.M.
      • Di Pietro A.
      • Barron D.
      • Ravelo A.G.
      • Castanys S.
      • Gamarro F.
      Multidrug resistance phenotype mediated by the P-glycoprotein-like transporter in Leishmania: a search for reversal agents.
      ), LiABCG4 (
      • Castanys-Muñoz E.
      • Alder-Baerens N.
      • Pomorski T.
      • Gamarro F.
      • Castanys S.
      A novel ATP-binding cassette transporter from Leishmania is involved in transport of phosphatidylcholine analogues and resistance to alkyl-phospholipids.
      ), and LiABCG6 (
      • Castanys-Muñoz E.
      • Pérez-Victoria J.M.
      • Gamarro F.
      • Castanys S.
      Characterization of an ABCG-like transporter from the protozoan parasite Leishmania with a role in drug resistance and transbilayer lipid movement.
      ) have also been shown to mediate MLF transport in Leishmania and have been implicated in MLF resistance. Thus, alterations in parasite and host cell drug transport could modify the antileishmanial effect of MLF. The purpose of this study was to identify the active carriers involved in transport and accumulation of miltefosine in human macrophages and to characterize their impact on intracellular drug-mediated parasite killing.

      Experimental Procedures

      Ethics Statement

      This study was approved and monitored by the institutional review board for ethical conduct of research involving human subjects of the Centro Internacional de Entrenamiento e Investigaciones Médicas in accordance with national (resolution 008430, República de Colombia, Ministry of Health, 1993) and international (Declaration of Helsinki and amendments, World Medical Association, Fortaleza, Brazil, October 2013) guidelines. All individuals voluntarily participated in the study, and written informed consent was obtained from each participant.

      Study Design and Study Subjects

      This study was designed to identify MLF membrane transporters in human macrophages and to evaluate their effect in the antileishmanial activity of MLF, aiming to define host cell factors that alter drug exposure of intracellular Leishmania. To identify candidate molecules with putative MLF transport function, we profiled gene expression of membrane transporters modulated by Leishmania infection and exposure to MLF in primary macrophages from healthy individuals. Modulation of gene expression was confirmed in primary macrophages from CL patients. Functional validation was performed using shRNA gene knockdown in THP-1 monocytes. MLF transport function was assessed by measurement of intracellular drug concentrations, cytotoxicity, and intracellular parasite killing assays.
      Adult patients (n = 19) with parasitologically confirmed diagnosis of active CL with a time of evolution <6 months, 18–65 years of age, and without apparent immune deficiencies (negative HIV test, no evidence of immunological disorder, nor treatment with medication having immunomodulating effects) participated in this study. Peripheral blood samples were obtained prior to initiation of antileishmanial treatment.

      Reagents and Chemicals

      MLF (Cayman Chemical Company, catalog no. 63280, lot no. 0410809-56) was dissolved in sterile DMSO following the manufacturer’s instructions (50 mg/62,5 ml DMSO) and stored at −20 °C until use. When indicated, MLF solutions were also prepared in PBS and used immediately after preparation. Working solutions were prepared in RPMI culture medium from the DMSO or PBS-dissolved stocks. Phorbol 12-myristate 13-acetate (PMA) was obtained from Sigma-Aldrich.

      Cell Culture and Differentiation

      Primary Macrophages

      Peripheral blood samples were taken from study participants, and mononuclear cells (PBMCs) were obtained by separation using a Ficoll-Hypaque (Sigma-Aldrich) gradient, following the manufacturer's instructions. Macrophages were differentiated from PBMCs by adherence to cell culture plasticware as previously described (
      • Gómez M.A.
      • Navas A.
      • Márquez R.
      • Rojas L.J.
      • Vargas D.A.
      • Blanco V.M.
      • Koren R.
      • Zilberstein D.
      • Saravia N.G.
      Leishmania panamensis infection and antimonial drugs modulate expression of macrophage drug transporters and metabolizing enzymes: impact on intracellular parasite survival.
      ).

      THP-1 Cell Line

      The human pro-monocytic cell line THP-1 and derived cell lines were maintained at 1 × 106 cells/ml in RPMI 1640 (Gibco) supplemented with 10% heat-inactivated FBS, 100 μg/ml streptomycin, and 100 units/ml penicillin at 37 °C and 5% CO2. Cultured cells were differentiated using 250 ng/ml PMA for 3 h and washed with PBS, and adherence was allowed for 24 h.

      Parasites, Infection, and Intracellular Parasite Survival Assays

      MLF-susceptible L. (Viannia) panamensis promastigotes (EC50 = 2.9 μm; 1.2 μg/ml (
      • Obonaga R.
      • Fernández O.L.
      • Valderrama L.
      • Rubiano L.C.
      • Castro Mdel M.
      • Barrera M.C.
      • Gomez M.A.
      • Gore Saravia N.
      Treatment failure and miltefosine susceptibility in dermal leishmaniasis caused by Leishmania subgenus Viannia species.
      )) stably transfected with the luciferase reporter gene (L.p-LUC001) and MLF nonsusceptible L. V. panamensis (L.p-LUC056; EC50 > 32 μm; >13.0 μg/ml, (
      • Obonaga R.
      • Fernández O.L.
      • Valderrama L.
      • Rubiano L.C.
      • Castro Mdel M.
      • Barrera M.C.
      • Gomez M.A.
      • Gore Saravia N.
      Treatment failure and miltefosine susceptibility in dermal leishmaniasis caused by Leishmania subgenus Viannia species.
      )) were kept at 25 °C in RPMI supplemented with 10% heat-inactivated FBS and 5 mg/ml hemin. L.p-LUC001 was maintained with additional 120 μg/ml Geneticin and L.p-LUC056 cultured in the presence of 60 μm MLF and 160 μg/ml Geneticin. Macrophages were infected stationary phase promastigotes opsonized with human AB+ serum at a 10:1 Leishmania-macrophage ratio for 2 h, washed twice with PBS, and incubated for 24 h at 34 °C 5% CO2. After infection was established, the cells were exposed for 24 h to MLF (4–16 μm) or left untreated for additional 24 h. Intracellular parasite survival was measured by luciferase activity (
      • Roy G.
      • Dumas C.
      • Sereno D.
      • Wu Y.
      • Singh A.K.
      • Tremblay M.J.
      • Ouellette M.
      • Olivier M.
      • Papadopoulou B.
      Episomal and stable expression of the luciferase reporter gene for quantifying Leishmania spp. infections in macrophages and in animal models.
      ).

      Drug Transporter Quantitative PCR Arrays and Gene Expression Profiling

      Screening of drug transporter gene expression was conducted as previously described (
      • Gómez M.A.
      • Navas A.
      • Márquez R.
      • Rojas L.J.
      • Vargas D.A.
      • Blanco V.M.
      • Koren R.
      • Zilberstein D.
      • Saravia N.G.
      Leishmania panamensis infection and antimonial drugs modulate expression of macrophage drug transporters and metabolizing enzymes: impact on intracellular parasite survival.
      ). The drug transporter Quantitative reverse transcriptase PCR array used was PAHS-070Z (SABiosciences-Qiagen). Selection of candidate genes was based on up- or down-regulation (±1.5-fold) following Leishmania infection and exposure to MLF at 16 μm. Corroboration of expression of selected genes was conducted by quantitative RT-PCR in macrophages from CL patients (n = 19) using TaqMan® gene expression assays (Applied Biosystems): abcb1 (P-glycoprotein, Hs01070641_g1), abca3 (Hs00975530_m1), and gapdh (Hs99999905_m1). Reactions were run on a Bio-Rad CFX-96 detection platform. Ct values were normalized to gapdh, and gene expression was calculated by ΔΔCt method and expressed as Log2 fold change.

      shRNA-mediated Transporter Gene Silencing in Host THP-1 Cells

      A lentivirus-based system was used for shRNA-mediated gene silencing in THP-1 monocytes as previously described (
      • Zhou H.
      • DeLoid G.
      • Browning E.
      • Gregory D.J.
      • Tan F.
      • Bedugnis A.S.
      • Imrich A.
      • Koziel H.
      • Kramnik I.
      • Lu Q.
      • Kobzik L.
      Genome-wide RNAi screen in IFN-γ-treated human macrophages identifies genes mediating resistance to the intracellular pathogen Francisella tularensis.
      ). Four independent sets of lentiviral particles for shRNA gene knockdown of ABCA3 were purchased from Sigma-Aldrich (SHCLNV-NM_001089: TRCN0000303547, TRCN0000315649, TRCN0000315713, and TRCN0000315714) THP-1 monocytes were transduced at an average multiplicity of infection of five viral particles to one monocyte in medium containing 10 μg/ml Polybrene. Transduced cells were selected and maintained under puromycin pressure (5 μg/ml) for a minimum of 5 days. pLKO.1 (Addgene) empty vector transduced cells were used as negative control. Gene knockdown was assessed by quantitative RT-PCR prior to every experimental procedure and considered acceptable when knockdown was >50%, compared with empty vector transfected THP-1 cells. Gene knockdown was also confirmed at the protein level by flow cytometry. Briefly, 1 × 106 monocytes were collected and washed with cold PBS and fixed with 0.5% formaldehyde for 30 min at 4 °C. The cells were permeabilized with 0.2% PBS-Tween 20 for 15 min at 37 °C, washed, and incubated with anti-human ABCA3 (Santa Cruz Biotechnology sc-134560) at a 1:50 ratio in FACS buffer containing 50% human AB+ serum for 1 h at 4 °C. The cells were then washed and incubated for 30 min with FITC-labeled anti-rabbit antibody (Jackson ImmunoResearch). Staining was measured on a BD-Accuri C6 cytometer. A total of 50.000 events were acquired and analyzed in FlowJo Vx software (Tree Star).

      Quantification of Intracellular MLF in THP-1 Cells

      PMA-differentiated empty vector control and ABCA3 knockdown (ABCA3KD) THP-1 cells were infected with L. V. panamensis (or left uninfected) and exposed for 24 h to [14C]MLF (kindly donated by Paladin Labs Inc.). Afterward, macrophages were washed three times with cold PBS-BSA (1%) to remove surface bound [14C]MLF. The cells were collected and centrifuged at 800 × g for 5 min, and the remaining pellet was resuspended in 50 μl of PBS. Intracellular MLF was quantified by scintillation counting measured in a Chameleon multiplate reader (Hidex) and documented as cpm. The number of cpm was converted to concentration and quantified using a linear calibration curve with a range of 0.25–128 μm MLF.

      Quantification of Intracellular MLF in Primary Macrophages

      Primary macrophages from CL patients were exposed in vitro to MLF (16 μm and 32 μm) for 24 h, washed with PBS-BSA (1%), and resuspended as described above. Intracellular MLF was quantified using a recently developed and validated LC-MS/MS method (
      • Kip A.E.
      • Rosing H.
      • Hillebrand M.J.
      • Castro M.M.
      • Gomez M.A.
      • Schellens J.H.
      • Beijnen J.H.
      • Dorlo T.P.
      Quantification of miltefosine in peripheral blood mononuclear cells by high-performance liquid chromatography-tandem mass spectrometry.
      ). Briefly, macrophages were lysed with 62.5% methanol. The homogenized solution was evaporated and resuspended in plasma, after which MLF was quantified on a calibration curve prepared in blank human K-EDTA plasma. This concentration was then back-calculated to total amount of miltefosine in the cell pellet.

      Cytotoxicity Assays

      PMA-differentiated ABCA3KD and empty vector control cells, and primary human macrophages were exposed to a dose range of MLF for 24 h. Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (ATCC).

      Phagocytosis Assays

      THP-1 cell lines were cultured and differentiated in 12-well plates at 0.35 × 106 cells/well in complete RPMI. The cells were incubated with 1 μl of stock solution of 1.0-micron fluorescent latex beads (Fluoresbrite Carboxilate YG 1.00 μm, catalog no. 15702, Polysciences, Inc.) for 2, 24, and 48 h. Adherent cells were detached with trypsin-EDTA, washed with PBS, and collected for FACS. A total of 20,000 events were acquired and analyzed.

      Confocal Microscopy

      PMA-differentiated THP-1 cells and primary human macrophages were plated on glass slides in 24-well plates, infected with L. V. panamensis transfected with green fluorescent protein and then exposed to 16 μm MLF or left untreated. The cells were washed, fixed with 4% formaldehyde, permeabilized with PBS containing 0.05% Nonidet P-40 (Igepal) and 1% BSA for 5 min, and blocked with 5% skim milk in PBS. Subsequently the cells were incubated with anti ABCA3 antibody for 1 h in a humid chamber, washed twice with PBS, and incubated with Alexa Fluor 568 donkey anti-rabbit IgG (Molecular Probes, Invitrogen) for 1 h. The slides were mounted and analyzed on a Zeiss Observer Z1 microscope and images captured using Zen 2009 software.

      Statistical Analysis

      D’Agostino and Pearson’s omnibus test (
      • D'Agostino R.
      • Pearson E.S.
      Tests for departure from normality: empirical results for the distributions of b2 and b1.
      ) was used to test for departures of quantitative data from normal distributions. Differences in gene expression were tested with one-way analysis of variance and Tukey’s multiple comparisons test. Differences in variance for the remaining experiments were analyzed with unpaired t test. Statistical dependence of variables was determined by Spearman’s rank correlation. A significance level of p < 0.05 was used for all statistical tests. Statistical analysis was performed using GraphPad Prism software (version 5).

      Discussion

      The effect of antimicrobials that target intracellular pathogens is, among others, determined by the capacity of drugs to be internalized into host cells and to traffic to the intracellular compartment in which pathogens reside. Our group and others have shown that killing of intracellular Leishmania by in vitro exposure to antimonial drugs is affected by the nature of the host cell (
      • Seifert K.
      • Escobar P.
      • Croft S.L.
      In vitro activity of anti-leishmanial drugs against Leishmania donovani is host cell dependent.
      ) and by differential expression and function of ABC transporters (
      • Gómez M.A.
      • Navas A.
      • Márquez R.
      • Rojas L.J.
      • Vargas D.A.
      • Blanco V.M.
      • Koren R.
      • Zilberstein D.
      • Saravia N.G.
      Leishmania panamensis infection and antimonial drugs modulate expression of macrophage drug transporters and metabolizing enzymes: impact on intracellular parasite survival.
      ,
      • Mookerjee Basu J.
      • Mookerjee A.
      • Banerjee R.
      • Saha M.
      • Singh S.
      • Naskar K.
      • Tripathy G.
      • Sinha P.K.
      • Pandey K.
      • Sundar S.
      • Bimal S.
      • Das P.K.
      • Choudhuri S.K.
      • Roy S.
      Inhibition of ABC transporters abolishes antimony resistance in Leishmania infection.
      ). In this study, we explored the role of human macrophages in the transport, accumulation, and exposure of intracellular Leishmania to MLF, the only oral drug available for the treatment of visceral and cutaneous leishmaniasis. Our results demonstrate the participation of ABCA3 in the transport of MLF across human macrophage membranes.
      Gene expression profiles have been one of the most powerful tools to understand the mechanisms of action of bioactive compounds, the cellular response to drugs, and the identification of therapeutic targets and mechanisms of resistance (
      • Cohen A.L.
      • Soldi R.
      • Zhang H.
      • Gustafson A.M.
      • Wilcox R.
      • Welm B.E.
      • Chang J.T.
      • Johnson E.
      • Spira A.
      • Jeffrey S.S.
      • Bild A.H.
      A pharmacogenomic method for individualized prediction of drug sensitivity.
      ,
      • Bosch T.M.
      Pharmacogenomics of drug-metabolizing enzymes and drug transporters in chemotherapy.
      ). Profiling the expression of membrane transporters modulated by L. V. panamensis infection and exposure to MLF suggested a possible contribution of ABCA1, ABCA3, and ABCD1 lipid transporters in MLF trafficking within human macrophages and supported the previously recognized role of ABCB1 in MLF efflux (
      • Rybczynska M.
      • Liu R.
      • Lu P.
      • Sharom F.J.
      • Steinfels E.
      • Di Pietro A.
      • Spitaler M.
      • Grunicke H.
      • Hofmann J.
      MDR1 causes resistance to the antitumour drug miltefosine.
      ). Although two solute liquid carriers, SLC7A11 and SLC3A1, were also induced by exposure to miltefosine, higher interindividual variability was observed and thus not selected for further functional validation.
      ABCA3 gene knockdown in THP-1 cells showed that MLF is a substrate of this membrane transporter. ABCA3 is a cholesterol and phosphatidylcholine influx transporter (
      • Matsumura Y.
      • Sakai H.
      • Sasaki M.
      • Ban N.
      • Inagaki N.
      ABCA3-mediated choline-phospholipids uptake into intracellular vesicles in A549 cells.
      ,
      • Cheong N.
      • Zhang H.
      • Madesh M.
      • Zhao M.
      • Yu K.
      • Dodia C.
      • Fisher A.B.
      • Savani R.C.
      • Shuman H.
      ABCA3 is critical for lamellar body biogenesis in vivo.
      ,
      • Chapuy B.
      • Koch R.
      • Radunski U.
      • Corsham S.
      • Cheong N.
      • Inagaki N.
      • Ban N.
      • Wenzel D.
      • Reinhardt D.
      • Zapf A.
      • Schweyer S.
      • Kosari F.
      • Klapper W.
      • Truemper L.
      • Wulf G.G.
      Intracellular ABC transporter A3 confers multidrug resistance in leukemia cells by lysosomal drug sequestration.
      ) found in the membranes of lysosomes and lamellar bodies, and its function has been primarily related to surfactant lipid metabolism and lamellar body biogenesis (
      • Cheong N.
      • Madesh M.
      • Gonzales L.W.
      • Zhao M.
      • Yu K.
      • Ballard P.L.
      • Shuman H.
      Functional and trafficking defects in ATP binding cassette A3 mutants associated with respiratory distress syndrome.
      ,
      • Cheong N.
      • Zhang H.
      • Madesh M.
      • Zhao M.
      • Yu K.
      • Dodia C.
      • Fisher A.B.
      • Savani R.C.
      • Shuman H.
      ABCA3 is critical for lamellar body biogenesis in vivo.
      ,
      • Ban N.
      • Matsumura Y.
      • Sakai H.
      • Takanezawa Y.
      • Sasaki M.
      • Arai H.
      • Inagaki N.
      ABCA3 as a lipid transporter in pulmonary surfactant biogenesis.
      ). Mutations in this transporter have been associated with surfactant dysfunction and respiratory diseases, especially in children (
      • Agrawal A.
      • Hamvas A.
      • Cole F.S.
      • Wambach J.A.
      • Wegner D.
      • Coghill C.
      • Harrison K.
      • Nogee L.M.
      An intronic ABCA3 mutation that is responsible for respiratory disease.
      ) and recently its expression in lung cells has been reported to modulate susceptibility to cisplatin and paclitaxel (
      • Overbeck T.R.
      • Hupfeld T.
      • Krause D.
      • Waldmann-Beushausen R.
      • Chapuy B.
      • Güldenzoph B.
      • Aung T.
      • Inagaki N.
      • Schöndube F.A.
      • Danner B.C.
      • Truemper L.
      • Wulf G.G.
      Intracellular ATP-binding cassette transporter A3 is expressed in lung cancer cells and modulates susceptibility to cisplatin and paclitaxel.
      ). Increased ABCA3 expression in mononuclear cells during acute myeloid leukemia has been shown to mediate chemoresistance by intravesicular drug sequestration (
      • Chapuy B.
      • Koch R.
      • Radunski U.
      • Corsham S.
      • Cheong N.
      • Inagaki N.
      • Ban N.
      • Wenzel D.
      • Reinhardt D.
      • Zapf A.
      • Schweyer S.
      • Kosari F.
      • Klapper W.
      • Truemper L.
      • Wulf G.G.
      Intracellular ABC transporter A3 confers multidrug resistance in leukemia cells by lysosomal drug sequestration.
      ). The predominant plasma membrane localization of ABCA3 in macrophages, coupled to increased drug accumulation in the ABCA3KD line and the inverse correlation between ABCA3 gene expression and intracellular MLF accumulation, suggests a function of ABCA3 as an efflux transporter in human macrophages. Following L. V. panamensis infection, ABCA3 was relocalized to intracellular compartments, potentially lysosomes as previously described (
      • Chapuy B.
      • Koch R.
      • Radunski U.
      • Corsham S.
      • Cheong N.
      • Inagaki N.
      • Ban N.
      • Wenzel D.
      • Reinhardt D.
      • Zapf A.
      • Schweyer S.
      • Kosari F.
      • Klapper W.
      • Truemper L.
      • Wulf G.G.
      Intracellular ABC transporter A3 confers multidrug resistance in leukemia cells by lysosomal drug sequestration.
      ). Whether recruitment of this transporter to vesicular membranes promotes its delivery to the phagosomal membrane remains to be validated.
      Unexpectedly, ABCA3 knockdown did not result in a significant difference in drug-mediated parasite killing of an MLF-susceptible L. V. panamensis strain. After 24 h of exposure to extracellular MLF concentrations as low as 4 μm, average intracellular concentrations were 3.3 and 5 μm in empty vector control and ABCA3KD THP-1 cells, respectively. This, together with the low EC50 of the MLF-susceptible strain (2.9 μm; 1.2 μg/ml (
      • Obonaga R.
      • Fernández O.L.
      • Valderrama L.
      • Rubiano L.C.
      • Castro Mdel M.
      • Barrera M.C.
      • Gomez M.A.
      • Gore Saravia N.
      Treatment failure and miltefosine susceptibility in dermal leishmaniasis caused by Leishmania subgenus Viannia species.
      )), suggests that the intracellular drug concentrations achieved in either control or ABCA3 KD macrophages are sufficient to kill MLF-susceptible parasites. In contrast, the nonsusceptible L. V. panamensis strain used in this study has been shown to have an EC50 value of >32 μm (
      • Obonaga R.
      • Fernández O.L.
      • Valderrama L.
      • Rubiano L.C.
      • Castro Mdel M.
      • Barrera M.C.
      • Gomez M.A.
      • Gore Saravia N.
      Treatment failure and miltefosine susceptibility in dermal leishmaniasis caused by Leishmania subgenus Viannia species.
      ). The observed ∼40% reduction in parasite load in ABCA3KD cell in the presence of 16 μm extracellular MLF is consistent with intracellular MLF concentrations that are toxic even to nonsusceptible strains achieved in ABCA3KD cells. This indicates that increased drug accumulation within host macrophages could be exploited as a mechanism to partially revert nonsusceptibility phenotypes.
      The role of ABCA3 during infection may not be restricted to MLF transport. ABCA3 knockdown decreased Leishmania uptake, potentially linked to reduced phagocytosis in ABCA3KD cells. Interestingly, Ced-7, a homologue of ABCA3 in Caenorhabditis elegans, functions in the engulfment of cell corpses during programmed cell death (
      • Wu Y.C.
      • Horvitz H.R.
      The C. elegans cell corpse engulfment gene ced-7 encodes a protein similar to ABC transporters.
      ). Alterations in lipid transport could affect cell membrane composition, indirectly impacting on cell membrane-dependent processes such as endocytosis and phagocytosis (
      • Tarling E.J.
      • de Aguiar Vallim T.Q.
      • Edwards P.A.
      Role of ABC transporters in lipid transport and human disease.
      ). Thus, reduced parasite burden in ABCA3KD macrophages could be mediated by an additive effect of decreased Leishmania internalization and increased intracellular MLF accumulation and drug-mediated parasite killing.
      Identification of a role for ABCA3 in reducing the antileishmanial activity of miltefosine raises the possibility of inhibiting ABCA3 to improve treatment success. Inhibition of ABCA3 expression has been reported to occur upon exposure of acute myeloid leukemia cells to the nonsteroidal anti-inflammatory drug indomethacin (
      • Song J.H.
      • Kim S.H.
      • Kim H.J.
      • Hwang S.Y.
      • Kim T.S.
      Alleviation of the drug-resistant phenotype in idarubicin and cytosine arabinoside double-resistant acute myeloid leukemia cells by indomethacin.
      ). The immunomodulatory properties of indomethacin make it unsuitable as component of Leishmania therapy, but a better understanding of how it affects ABCA3 expression may enable discovery of other, more suitable repressors.
      We have previously reported an inverse correlation between Leishmania ABCA3 gene expression in amastigotes and MLF susceptibility, supporting the possibility that higher expression of vesicular ABCA3 in the parasite could promote susceptibility through increased drug accumulation (
      • Obonaga R.
      • Fernández O.L.
      • Valderrama L.
      • Rubiano L.C.
      • Castro Mdel M.
      • Barrera M.C.
      • Gomez M.A.
      • Gore Saravia N.
      Treatment failure and miltefosine susceptibility in dermal leishmaniasis caused by Leishmania subgenus Viannia species.
      ). Although not functionally validated, this hypothesis would suggest that ABCA3 inhibitors could induce drug resistance in the parasite. However, the amino acid sequences of Leishmania ABCA3 (retrieved from either L.V. braziliensis, Leishmania major, or Leishmania infantum genomes) and mammalian ABCA3 share only 30% sequence identity; this divergence supports targeted intervention of host ABCA3. Conceivably, pharmacological modulation of host ABCA3 and potentially other ABC lipid transporters could potentiate the antileishmanial effect of miltefosine for the treatment of infections caused by Leishmania strains of diverse drug susceptibilities.
      Here we report the expression of ABCA3 in human macrophages, its role in the transport of miltefosine, and its modulation during exposure to miltefosine. Our results provide evidence in support of the central role of host cell drug transport and accumulation in the direct activity of drugs that target intracellular pathogens. Characterization of the molecular mechanisms that mediate host cell drug influx, efflux, and trafficking between intracellular compartments increases our understanding of the pharmacokinetic-pharmacodynamic relationships of drugs active against intracellular pathogens. More importantly, it allows for identification of novel host-directed therapeutic strategies to optimize intracellular exposure and thus efficacy of both available and future antileishmanial drugs.

      Author Contributions

      L. C. T. D. performed, analyzed, and interpreted the data shown in Figs. 3, 5, and 7. A. N. performed and analyzed the experiments shown in Figs. 2, 4, and 6 and in Table 1. D. J. G. conceived and analyzed experiments using ABCA3 knockdown cells. A. K. performed and analyzed intracellular MLF quantitation assays. T. P. C. D. analyzed and interpreted data on intracellular drug transport and measurement of drug concentrations. D. A. V. performed and analyzed the experiments shown in Figs. 1FIGURE 2, FIGURE 3, 5, and 7. M. A. G. conceived and coordinated the study and assembled the paper. All authors actively participated in writing and revising all versions of the paper, reviewed the results, and approved the final version of the manuscript.

      Acknowledgments

      We gratefully acknowledge the patients and volunteers who participated in this study and the members of the Clinical Unit of the Centro Internacional de Entrenamiento e Investigaciones Médicas in Cali and Tumaco for recruitment of participants and follow-up. We thank Paladin Labs Inc. for donation 14C-radiolabeled miltefosine and BD Biosciences for donation of the Accuri C6 platform.

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