J Antimicrob Chemother 2014; 69: 139 – 149
doi:10.1093/jac/dkt334 Advance Access publication 24 August 2013
Leishmania panamensis infection and antimonial drugs modulate
expression of macrophage drug transporters and metabolizing
enzymes: impact on intracellular parasite survival
Maria Adelaida Gómez1*, Adriana Navas1, Ricardo Márquez1, Laura Jimena Rojas1, Deninson Alejandro Vargas1,
Victor Manuel Blanco1, Roni Koren2, Dan Zilberstein2 and Nancy Gore Saravia1
1
Centro Internacional de Entrenamiento e Investigaciones Médicas (CIDEIM), Carrera 125 No. 19 –225 Cali, Colombia; 2Technion Israel
Institute of Technology, Faculty of Biology, 32000 Haifa, Israel
*Corresponding author. Tel: +57-(2)-5552164; Fax: +57-(2)-5552638; E-mail: [email protected]
Received 10 May 2013; returned 22 June 2013; revised 8 July 2013; accepted 22 July 2013
Objectives: Treatment failure is multifactorial. Despite the importance of host cell drug transporters and metabolizing enzymes in the accumulation, distribution and metabolism of drugs targeting intracellular pathogens,
their impact on the efficacy of antileishmanials is unknown. We examined the contribution of pharmacologically
relevant determinants in human macrophages in the antimony-mediated killing of intracellular Leishmania panamensis and its relationship with the outcome of treatment with meglumine antimoniate.
Methods: Patients with cutaneous leishmaniasis who failed (n¼8) or responded (n¼ 8) to treatment were
recruited. Gene expression profiling of pharmacological determinants in primary macrophages was evaluated
by quantitative RT–PCR and correlated to the drug-mediated intracellular parasite killing. Functional validation
was conducted through short hairpin RNA gene knockdown.
Results: Survival of L. panamensis after exposure to antimonials was significantly higher in macrophages from
patients who failed treatment. Sixteen macrophage drug-response genes were modulated by infection and exposure to meglumine antimoniate. Correlation analyses of gene expression and intracellular parasite survival revealed
the involvement of host cell metallothionein-2A and ABCB6 in the survival of Leishmania during exposure to antimonials. ABCB6 was functionally validated as a transporter of antimonial compounds localized in both the cell and
phagolysosomal membranes of macrophages, revealing a novel mechanism of host cell-mediated regulation of
intracellular drug exposure and parasite survival within phagocytes.
Conclusions: These results provide insight into host cell mechanisms regulating the intracellular exposure of
Leishmania to antimonials and variations among individuals that impact parasite survival. Understanding of
host cell determinants of intracellular pharmacokinetics/pharmacodynamics opens new avenues to improved
drug efficacy for intracellular pathogens.
Keywords: ABCB6, MT2A, host– pathogen interactions, leishmaniasis, phagolysosomes
Introduction
In the absence of a vaccine and sustainable strategies of vector
management, control of leishmaniasis relies on drug therapy.
Pentavalent antimonials [meglumine antimoniate (Glucantime)
or sodium stibogluconate (Pentostam)] continue to be the first-line
treatment in Latin America. However, the emergence of resistance
to antimonial drugs and increased rates of treatment failure
threaten this control measure.
Although treatment failure is commonly attributed to parasite
drug resistance, the therapeutic response is multifactorial, involving
intrinsic or acquired differences in the drug susceptibility of
the infecting Leishmania strain, the host immune response,
pharmacokinetic (PK) and pharmacodynamic characteristics of
the drugs, and genetic, metabolic and physiological variations
among individual hosts. The central role of immune functions in
the effectiveness of antimonials has been supported by studies in
murine models of visceral leishmaniasis and immunocompromised patients. In mice, deficiency in tumour necrosis factor-a,
interferon-g or interleukin-12 reduces the efficacy of antimonials,1 – 3 while in patients, down-regulation of these cytokines
has been linked to mucosal and chronic cutaneous disease,4,5
# The Author 2013. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
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139
Gómez et al.
and immunodeficiencies are conducive to higher rates of disease
reactivation following treatment.6 Despite this, failure is also frequently observed in immunocompetent individuals.
Our group and others have demonstrated that antimonial treatment failure in patients with cutaneous leishmaniasis (CL) can
occur in the absence of parasite drug resistance during infections
with drug-susceptible Leishmania.7 – 9 Furthermore, evidence suggests that the PK of antimony (Sb) in plasma alone does not
define treatment response, as we have shown minimal interindividual variation in the plasma PK of Sb in adult patients treated with
meglumine antimoniate.10 Therefore, other factors, such as intracellular PK and pharmacodynamics, are likely to play a key role in
the therapeutic response to antimonials. Despite this, understanding of the role of pharmacological determinants in the therapeutic
response is largely unexplored.
Leishmania, an obligate intracellular parasite, resides preferentially in phagocytes within membrane-defined phagolysosomes;
therefore, antileishmanial drugs must cross these host cell barriers
to target the intracellular parasite. Mookerjee Basu et al.11 showed
that monocytes from patients with visceral leishmaniasis that
were unresponsive to pentavalent antimonials, overexpress the
multidrug resistance protein-1 (MRP-1) and permeability glycoprotein (P-gp) at the plasma membrane and this phenotype correlated
with lower accumulation of intracellular Sb. However, the
molecules involved in the exposure of the intraphagosomal parasite to the drug and their influence on the drug-mediated intracellular parasite killing remain unknown.
Through the exploration of host –pathogen –drug interactions,
we sought to identify pharmacological determinants of human
macrophages that affect the Sb-mediated intracellular parasite
killing. Our results show that infection of human macrophages with
Leishmania and exposure to antimonial drugs modulate the expression of drug transporters and metabolizing enzymes in the host cell.
These findings expand the scope of this highly dynamic host–
pathogen interaction to the modulation of antileishmanial drug
exposure and response. We identified human ABCB6 as a novel transporter of antimonial compounds in human macrophages, located at
the cell membrane and surrounding the Leishmania-containing
phagosome. Importantly, the expression of ABCB6 in human macrophages correlates with intracellular parasite survival, hence identifying this molecule as an important host cell regulator of the
Sb-mediated parasite killing.
Methods
Ethics
This study was approved and monitored by the institutional review board for
ethical conduct of research involving human subjects of the Centro
Table 1. Characteristics of patients and Leishmania strains
Characteristics
Treatment response
Treatment failure
Subjects
age (years), mean (range)
male, n (%)
ethnic group, n (%)
Afro-Colombian
Mestizo
lesions per subject, median (range)
duration of older lesion (months), median (range)
n¼8
40.3 (24 –60)
5 (62.5)
n¼8
38.2 (20 –50)
7 (87.5)
8 (100)
0
1.5 (1–7)
1 (1– 2)
6 (75)
2 (25)
1 (1– 3)
2 (1– 4)
Lesion characteristics
location of lesion, n (%)
upper limbs
head and neck
trunk
lower limbs
type of lesion, n (%)
ulcer
plaque
other
lesion areab (mm2), median (range)
n¼19
n¼12
11 (57.9)
4 (21.1)
0
4 (21.1)
7 (58.3)
1 (8.3)
4 (33.3)
0
19 (100)
0
0
26.6 (9.4 –62.8)
10 (83.3)
1 (8.3)
1 (8.3)
63.7 (20.5– 111.4)
n¼8
n¼8
8 (100)
0
7 (87.5)
1 (12.5)
8 (100)
0
8 (100)
0
Leishmania strains
species identification, n (%)
Leishmania (Viannia) panamensis
Leishmania (Viannia) guyanensis
susceptibility profile, n (%)
susceptible
resistant
a
P valuea
0.368
0.56
0.46
0.26
0.136
0.023
0.16
0.0001
0.46
Statistical analyses for categorical variables were performed using Fisher’s exact test and for continuous variables using the Mann–Whitney U-test.
Significance was established as P, 0.05.
b
Lesions were not measured in two subjects, one in each group.
140
Leishmania and Sb modulate host drug response
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, Seoul, Korea, October 2008) guidelines. All individuals
voluntarily participated in the study and written informed consent was
obtained from each patient.
Study design and study subjects
In order to evaluate the role of host cell factors on the therapeutic outcome
of CL, patients were included based on the therapeutic response to meglumine antimoniate, infection with a Leishmania strain that was susceptible
to Sb, age of 18 –65 years and no apparent immune deficiencies or negative
HIV tests. This strategy minimized the likelihood of potentially confounding
effects of treatment failure as a result of parasite drug resistance, variations
in drug PK10 or altered immune functions. The drug susceptibility of the clinical strains isolated from the participating patients was determined as intracellular amastigotes as previously described.12 Gene expression profiling of
human drug transporters and metabolizing enzymes of primary human
macrophages derived from peripheral blood mononuclear cells (PBMCs)
obtained from each patient was employed to identify host cell genes with
putative roles in intracellular parasite survival during drug exposure. The
human monocytic cell line THP-1 was used for functional validation assays.
Adult patients (18 –65 years) with diagnosis of active CL or documented
history of parasitologically confirmed CL were recruited for this study based
on the following criteria: (i) time of evolution ,6 months; (ii) availability of
the corresponding Leishmania strain; (iii) lack of apparent immune deficiencies (negative HIV test, evidence of immunological disorder or treatment
with medication having immunomodulating effects); and (iv) who received
standard-of-care treatment with meglumine antimoniate (20 mg/kg/day
for 20 days). Patients were included in two study groups: (i) those failing
standard-of-care treatment with meglumine antimoniate (n¼8); and (ii)
those who were cured (n¼8).
Treatment outcome was evaluated at week 13 after initiation of treatment. Cure was defined as complete re-epithelialization and absence of inflammatory signs for all lesions. Clinical failure was defined as: incomplete
re-epithelialization and/or the presence of induration, raised borders or
redness in any lesion; reactivation of the original lesion(s); or the appearance of new lesions. The characteristics of the study participants are summarized in Table 1.
Reagents and chemicals
Additive-free meglumine antimoniate (Walter Reed 214975AK; lot no.
BLO918690-278-1A1W601) was kindly provided by the Walter Reed Army
Institute, Silver Spring, MD, USA. Phorbol-12-myristate 13-acetate (PMA)
was obtained from Sigma– Aldrich.
Clinical strains and drug susceptibility assays
Leishmania were isolated by needle aspiration of cutaneous lesions. Positive
cultures were typed using monoclonal antibodies. The drug susceptibility of
the intracellular parasites was estimated by evaluation of the percentage
parasite survival in PMA-differentiated U-937 macrophages after exposure
to meglumine antimoniate (32 mg/mL Sb; screening concentration), compared with the control without drug exposure. Leishmania strains were
defined as Sb susceptible and Sb resistant when the percentage survival
after drug exposure was ,30% and .70%, respectively12 (Table S1, available as Supplementary data at JAC Online).
Cell culture and differentiation
Peripheral blood samples from healthy volunteers or patients with CL were
obtained and processed to obtain PBMCs using a Ficoll-Hypaque (Sigma–
JAC
Aldrich) gradient, following the manufacturer’s instructions. Macrophages
were differentiated from PBMCs by adherence to cell culture plasticware
in serum-free RPMI for 2 h, followed by 7 days of incubation in RPMI supplemented with 20% fetal bovine serum (FBS) at 378C and 5% CO2. The human
monocytic cell line THP-1 was maintained at 1×106 cells/mL in RPMI 1640
(Gibco) supplemented with 10% heat-inactivated FBS, 100 mg/mL
streptomycin and 100 U/mL penicillin at 378C and 5% CO2. Cultured cells
were differentiated to macrophages using 300 ng/mL PMA for 3 h,
washed with PBS and allowed to adhere for 24 h.
Parasites, infection and intracellular survival assays
Sb-susceptible Leishmania panamensis MHOM/CO/2002/3594 promastigotes were stably transfected with the luciferase reporter gene (L.p-LUC
001) cloned into the pGL2-a-NEO-a vector as previously described.13
L. panamensis MHOM/CO/1995/1989/GFP012 was transfected with the
green fluorescent protein (GFP) reporter gene cloned into the
pGEM7zf-a-NEO-a vector14 (L.p-GFP). Promastigotes were kept at 258C in
RPMI supplemented with 10% heat-inactivated FBS, 5 mg/mL haemin and
.120 mg/mL geneticin. Macrophages were infected with stationary-phase
promastigotes at a 10:1 Leishmania:macrophage ratio for 2 h and washed
twice with PBS, followed by a 24 h chase period at 348C and 5% CO2. Nonphagocytized parasites were removed by washing with PBS, followed by
the addition of meglumine antimoniate (8–32 mg/mL Sb). Intracellular parasite survival was measured by luciferase activity as previously described.13
Drug transporter and metabolizing enzyme PCR arrays
and gene expression profiling
Total RNA was extracted from cultured cells with Trizol reagent (Invitrogen,
USA) followed by RNA cleanup with RNeasy Mini Kit columns (Qiagen, USA).
The expression of drug transporter and drug-metabolizing enzyme genes
was evaluated by using commercially available PCR arrays (catalogue
numbers PAHS-070Z and PAHS-002Z, respectively; SABiosciences-Qiagen)
following the manufacturer’s instructions. The data were analysed using
the RT2 Profiler PCR Array Data Analysis online tool provided by the manufacturer. Selection of the candidate genes was based on up- or down-regulation
(+2-fold) following Leishmania infection and exposure to meglumine antimoniate at 32 mg/mL Sb in two independent expression array replicas of
infected and drug-treated human macrophages. Validation of the gene
expression profiles was conducted by quantitative RT–PCR (qRT–PCR).
Briefly, PBMC-derived macrophages from patients with CL were infected
with L.p-LUC 001 as described above, followed by 24 h of exposure to meglumine antimoniate (32 mg/mL Sb). Cells were collected, RNA extracted and
RNA reverse transcribed with a high-capacity cDNA reverse transcription kit
(Applied Biosystems). Expression of genes abcb1 (P-gp, Hs01070641_g1),
abcb6 (Hs01039213_m1), abcc1 (MRP-1, Hs01561510_m1), abcc2
(Hs00166123_m1), slc7a11 (Hs00204928_m1), slc5a4 (Hs00429526_m1),
aqp-1 (Hs00166067_m1), aqp-9 (Hs01035887_m1), mt2a (Hs01591333_g1),
abp1 (Hs00175631_m1), alox5 (Hs01095330_m1), nos3 (Hs01574659_m1),
mgst2 (Hs00182064_m1), gsr (Hs00167317_m1), gstm3 (Hs00356079_m1),
pkm2 (Hs00762869_s1) and gapdh (Hs99999905_m1) was evaluated using
TaqMan Gene Expression Assays (Applied Biosystems) and a Bio-Rad CFX-96
real-time PCR detection platform. Ct values were normalized to GAPDH. Gene
expression was calculated by the DDCt method and expressed as 22DDCt.
Short hairpin RNA (shRNA)-mediated drug transporter
gene silencing in host macrophages
A lentivirus-based system was used for shRNA-mediated gene silencing in
THP-1 monocytes as previously described.15 Lentiviral particles for shRNA
gene knockdown of ABCB6 were generated as follows: three independent
sets of oligonucleotide pairs for gene knockdown of human abcb6,
slc7a11 and aqp-9 (Table S2, available as Supplementary data at JAC
141
Gómez et al.
Online) were synthesized and cloned into the pLKO.1-TCR cloning vector
(Addgene, Cambridge, MA, USA).16 Lentiviral particles were generated by
co-transfection of endotoxin-free hairpin-containing pLKO.1-TCR, psPAX2
and pMD2.G (Addgene) into HEK-293 T cells. FuGENE HD (Roche) was used
as the transfection agent. Lentivirus-containing cell supernatant was collected 4 days after transfection and subsequently used to transduce
THP-1 monocytes in medium containing 10 mg/mL polybrene. Transduced
cells were selected under puromycin pressure (5 mg/mL) for a minimum of
5 days. Gene knockdown was confirmed by qRT–PCR.
Quantification of intracellular Sb
PBMC-derived primary macrophages from CL patients were infected with
L. panamensis for 24 h, or left uninfected, followed by exposure to meglumine antimoniate at concentrations of 16 and 32 mg/mL Sb for an additional 24 h. Cells were washed three times with PBS to remove extracellular
traces of Sb. Macrophage samples were resuspended in 100 mL of concentrated nitric acid and stored at 2808C until processing. Fifty microlitres of
the concentrated sample was diluted into 7 mL of ultrapure water followed
by filtration through a 0.22 mm filter. The intracellular Sb concentration was
measured by inductively coupled plasma optical emission spectrometry in
an iCAP 6300 platform (Thermo Scientific) according to the manufacturer’’s
instructions for water analysis. Sb concentrations were calculated based on
a calibration curve with a linear range of 0.5– 5 mg/mL. Sb concentrations
were normalized to the total cell number in each sample preparation.
Confocal microscopy
Macrophages were plated on glass coverslips and infected with L.p-GFP for
2 h followed by a chase period of 24 h. Cells were washed with ice-cold PBS,
fixed with 4% formaldehyde at 48C and permeabilized for 5 min in PBS containing 1% BSA and 0.05% NP-40. After blocking in 5% non-fat evaporated
milk in PBS, the coverslips were incubated with rabbit anti-human a-ABCB6
polyclonal antibody (H-300, Santa Cruz Biotechnology). The slides were
washed with PBS and incubated with Alexa Fluor 568 a-rabbit antibody
(Molecular Probes). After mounting, the cells were visualized by confocal
microscopy with a Zeiss LSM 700 system.
Results
Survival of L. panamensis exposed to meglumine
antimoniate is significantly higher in macrophages from
patients who failed treatment
Sb-susceptible Leishmania strains were isolated from eight
patients with CL who responded to standard-of-care treatment
with meglumine antimoniate and from eight patients who failed
treatment (Table 1), ruling out treatment failure as a consequence
of parasite drug resistance. To understand the contribution of host
cell factors in the response to treatment in patients infected with
Sb-susceptible Leishmania, we evaluated the intracellular survival
of L. panamensis in primary macrophages when exposed to meglumine antimoniate. Survival of the Sb-susceptible L. panamensis
strain L.p-LUC 001 was significantly higher (P ¼ 0.004) in macrophages from patients who failed treatment (n¼ 8) (Figure 1a),
compared with macrophages from patients who responded to
treatment (n¼8). Parasite loads following 24 h of infection in the
absence of drug were similar in macrophages from the two study
groups (Figure 1b), indicating that the difference in survival following drug exposure was not due to increased infection of macrophages from non-responding patients.
142
Although Sb drug treatment failure in patients with visceral
leishmaniasis was previously reported to be linked with reduced
intracellular drug accumulation as a consequence of MRP-1 and
P-gp overexpression in monocytes,11 neither MRP-1 and P-gp
gene expression nor total intracellular Sb accumulation differed
in either infected or uninfected macrophages from CL patients
who failed or responded to meglumine antimoniate (Figure S1,
available as Supplementary data at JAC Online). This suggests
that other host cell mechanisms influence the antileishmanial efficacy of Sb and the intracellular parasite survival, despite intrinsic
susceptibility of the parasite to antimonials.
Infection with L. panamensis and exposure to meglumine
antimoniate modulate gene expression of macrophage
transporters and metabolizing enzymes
To understand the mechanisms contributing to the increased survival of Leishmania in macrophages from patients with antimonial
drug treatment failure, we sought to identify pharmacological
determinants in macrophages having potential to modulate the
exposure of the intraphagosomal parasite to the drug. The expression of 84 human drug transporter and 84 drug-metabolizing
enzyme genes was assessed by qRT –PCR in infected and meglumine antimoniate-treated primary human macrophages from
healthy donors using commercially available PCR arrays. A highly
dynamic host –parasite and host–drug interaction was revealed,
in which the expression of one-quarter of these genes was modulated (Table S3, available as Supplementary data at JAC Online), including macrophage ATP-binding cassette (ABC) transporters,
solute liquid carriers (SLCs), aquaporins (AQPs) and phase II metabolizing enzymes. The magnitude of host cell genes modulated
+2-fold by infection or infection followed by drug exposure was
similar (24 and 32 of 168 genes, respectively) (Table S3).
The consistency in the modulation of transporters of glutathione (GSH) and GSH conjugates (ABCB1, ABCC1 and ABCC2),
enzymes involved in GSH metabolism [GSH reductase (GSR), microsomal GSH S-transferase 2 (MGST2), GSH S-transferase mu 3
(GSTM3) and amiloride-binding protein 1 (ABP1)] and heavy
metal transporters and scavenger molecules [ABCB6 and AQP-9,
and metallothionein-2A (MT2A), respectively] suggested their
involvement in the transport and detoxification of antimonials in
human macrophages. We therefore selected these genes for
further validation by qRT–PCR in macrophages from patients
with CL.
A heat map representation of the fold change in the gene expression of infected and drug-exposed macrophages compared
with that in uninfected and untreated cells (Figure 2a) shows
that exposure to meglumine antimoniate had a greater effect on
host cell gene expression than L. panamensis infection. Meglumine
antimoniate strongly induced the expression of abcb6, slc7a11,
mt2a, gsr, gstm3 and to a lesser extent nos3 and abcc2, while
down-regulating alox5 and p-gp (P,0.05) (Figure 2b). Conversely,
infection with L. panamensis down-regulated aqp-1, mgst2, nos3,
abp1 and alox5 (P,0.05) and induced mt2a and slc7a11
(P,0.05). The effect on gene expression exerted by exposure
to antimonials was unaffected in infected cells, with the
exception of induction of nos3 and gstm3, which was abrogated
in L. panamensis and meglumine antimoniate-treated cells
(Figure 2b). The down-regulation of alox5 and induction of mt2a
JAC
Leishmania and Sb modulate host drug response
P = 0.004
(a)
2 × 1005
80
Relative light units
(27.5, 88)
100
Percentage survival
(b)
(11.7, 42.6)
60
40
20
0
L.p
L.p + MA
L.p + MA
Response
Failure
1 × 1005
6 × 1004
4 × 1004
2 × 1004
0
Response
Failure
Figure 1. Survival of L. panamensis (L.p) is significantly higher in macrophages from patients who failed meglumine antimoniate (MA) treatment. (a)
Intracellular survival of Sb-susceptible L. panamensis after in vitro exposure to meglumine antimoniate (32 mg/mL Sb) for 24 h in primary
macrophages of patients with CL who failed (n¼8) or responded (n¼ 8) to meglumine antimoniate treatment. (b) Intracellular parasite loads in
infected and untreated macrophages. Parasite survival was measured by luciferase activity. Data are shown as percentage survival compared with
infected macrophages unexposed to meglumine antimoniate (a) and as absolute relative light units in infected macrophages unexposed to the drug
(b). Statistical difference was determined by parametric or non-parametric analysis of variance tests, as appropriate.
exerted by L. panamensis and meglumine antimoniate was significantly potentiated in infected and drug-treated macrophages. No
significant differences were observed for mrp-1, aqp-9, slc5a4 and
pkm2 gene expression in macrophages from CL patients (Figure 2b).
abcb6 and mt2a gene expression inversely correlates
with intracellular survival of L. panamensis
To identify host cell transporter and metabolizing enzyme genes
with putative contributions in the Sb-mediated parasite killing,
we evaluated the correlation between host cell gene expression
profiles and Leishmania survival upon exposure to meglumine antimoniate in macrophages from CL patients. Of the panel of 16 genes
analysed, only expression of host cell abcb6 and mt2a were inversely correlated (P,0.05) with the intracellular survival of L. panamensis (Figure 3a), indicating that the up-regulation of abcb6
and mt2a induced by meglumine antimoniate promotes intracellular parasite killing. Outlier identification analysis using the ROUT
coefficient (Q¼ 1% and Q¼ 5%) did not identify any outlier data;
thus, the statistical significance of these correlations was substantiated. The expression of abcb6 was positively correlated with
mt2a, slc7a11, gsr and aqp-9 expression in infected and drugtreated cells, and mt2a was positively correlated with slc7a11,
gsr and abcb6 expression (Figure 3b). This suggests possible coregulatory networks to be operating in response to antimonial drug
exposure and Leishmania infection and putative functional implications for these gene products. No correlation between the expression of p-gp, mrp-1, abcc2, slc5a4, slc7a11, aqp-1, aqp-9,
mgst2, gstm3, gsr, abp1, pkm2, nos3 and alox5 and the intracellular
survival of L. panamensis was observed (data not shown).
Macrophage ABCB6 is localized in the cell and
phagolysosomal membranes in human macrophages
ABCB6 is a transporter of porphyrin17 and heavy metals including
arsenic (As),18 a metalloid closely related to Sb. ABCB6 has been
shown to localize to the mitochondrial and cell membranes of
glioblastoma and erythroleukaemia cell lines19 and to late endosomal/lysosomal compartments of red blood cells and liver
homogenates.17,20 – 22 However, its localization in myeloid cells
was unknown. To further dissect the contribution of ABCB6 in the
Sb-mediated parasite killing, we analysed its subcellular localization in human macrophages. Confocal microscopy of uninfected
and L. panamensis-infected THP-1 macrophages showed that
ABCB6 localizes to the cell membrane and in intracellular structures in uninfected macrophages (Figure 4a). In line with its previously shown subcellular localization at the cell membrane and
endolysosomal compartments, ABCB6 was found in the plasma
membrane of macrophages and in the phagosome surrounding
L. panamensis amastigotes after 24 h of infection (Figure 4a and b).
ABCB6 expression is strongly induced by Sb and transports
antimonial compounds in human macrophages
Having observed that abcb6 gene expression is strongly induced in
human macrophages following exposure to meglumine antimoniate (Figure 2b) at concentrations as low as 8 mg/mL (data not
shown) and that its expression inversely correlates with Leishmania
survival after exposure to antimonials, we sought to functionally
validate ABCB6 as an Sb transporter.
We first quantified the intracellular content of Sb in wild-type
(WT) THP-1 cells and evaluated the dynamics of accumulation.
The intracellular Sb concentration was linear (r 2 ¼ 0.99) over a
range of exposure concentrations from 8 to 32 mg/mL Sb
(Figure 5a). The average intracellular content of Sb was 50, 90
and 200 ng/106 cells when exposed for 24 h to Sb at 8, 16 and
32 mg/mL, respectively (Figure 5a). After single-dose exposure to
meglumine antimoniate at either 16 or 32 mg/mL Sb, maximum
intracellular Sb was detected at 24 h and 50% of the total intracellular Sb was retained after 72 h of exposure (Figure 5b). We
created a human monocytic THP-1 ABCB6-knockdown (ABCB6KD)
cell line, with stable gene knockdown ranging between 60% and
90%, as confirmed by qRT –PCR. A time-dependent Sb accumulation curve in ABCB6KD and WT macrophages showed a 12% –
27% increase in intracellular Sb accumulation in ABCB6KD when
exposed to 32 mg/mL Sb (WT AUC0 – 72 ¼10.5+2 mg/106 cells
and ABCB6KD AUC0 – 72 ¼11.8+1.1 mg/106 cells, respectively) or
143
Gómez et al.
(a)
+3
L.p
MA
0
L.p +
MA
Gene expression (2-DDct)
**
**
ABCC2
**
**
3
**
2.0
1.5
1.0
2
10
2
1
5
1
0
0
0
0.5
0.0
Gene expression (2-DDct)
AQP-9
3
AQP-1
**
20
3
15
2
2
10
1
1
5
0
0
0
**
3
2
SLC5A4
4
4
1
0
MT2A
**
Gene expression (2-DDct)
PKM2
NOS3
ABCB6
**
15
MGST2
ABP1
P-gp
ALOX5
3
**
AQP-1
AQP-9
SLC5A4
MRP-1
GSTM3
GSR
ABCC2
MRP-1
P-gp
**
2.5
ABCB6
(b)
SLC7A11
MT2A
–3
**
50
**
40
**
30
1.0
2
ABP1
**
6
1.5
**
**
8
2.0
**
**
PKM2
NOS3
**
**
4
**
SLC7A11
**
**
**
**
2
20
0.5
10
0
0.0
0
0
ALOX5
Gene expression (2-DDct)
144
4
MGST2
GSR
**
**
2.5
**
8
**
2.0
**
**
**
2
1.5
4
1.0
1
2
0.5
0
L.p
MA
**
**
**
6
2
**
3
**
**
**
GSTM3
–
–
+
–
–
+
+
+
0.0
L.p
MA
–
–
+
–
–
+
+
+
0
L.p
MA
–
–
+
–
–
+
+
+
0
L.p
MA
–
–
+
–
–
+
+
+
JAC
Leishmania and Sb modulate host drug response
P = 0.045
r = –0.43
8
6
4
2
Responders
Non-responders
40
MT2A expression (2-DDCt)
Responders
Non-responders
10
ABCB6 expression (2-DDCt)
(a)
P = 0.019
r = –0.52
30
20
10
0
0
0
20
40
60
80
Parasite survival (%)
100
0
20
40
60
80
Parasite survival (%)
100
5
0
0
10
5
0
0
3
2
1
0
0
2
4
6
8
10
ABCB6 expression (2-DDCt)
P = 0.0003
r = 0.76
15
10
20
30
40
MT2A expression (2-DDCt)
P = 0.006
r = 0.6
2
4
6
8
10
ABCB6 expression (2-DDCt)
P = 0.04
r = 0.42
4
3
2
1
0
0
10
20
30
40
MT2A expression (2-DDCt)
AQP-9 expression (2-DDCt)
10
4
ABCB6 expression (2-DDCt)
GSR expression (2-DDCt)
P = <0.0001
r = 0.80
15
GSR expression (2-DDCt)
SLC7A11 expression (2-DDCt)
SLC7A11 expression (2-DDCt)
(b)
2.5
P = 0.02
r = 0.5
2.0
1.5
1.0
0.5
0.0
0
2
4
6
8
10
ABCB6 expression (2-DDCt)
P = 0.04
r = 0.44
10
8
6
4
2
0
0
10
20
30
40
MT2A expression (2-DDCt)
Figure 3. Intracellular survival of L. panamensis is correlated with expression of macrophage abcb6 and mt2a. (a) Correlation analysis of intracellular
parasite survival versus gene expression of drug transporters and metabolizing enzymes and (b) correlation analysis of gene expression of
pharmacological determinants in infected and drug-treated (meglumine antimoniate; 32 mg/mL Sb) macrophages from CL patients who failed (n ¼8)
or responded (n¼8) to meglumine antimoniate treatment. Statistical correlation was assessed by the Spearman correlation test and significance was
established when P,0.05.
16 mg/mL Sb (WT AUC0 – 72 ¼ 5.2+0.1 mg/106 cells and ABCB6KD
AUC0 – 72 ¼ 6.62+0.2 mg/106 cells, respectively), substantiating
the function of ABCB6 as an Sb transporter.
ABCB6 promotes Sb-mediated killing of intracellular
Leishmania
To validate the role of host cell ABCB6 in the drug-mediated intracellular parasite killing, we evaluated the survival of L. panamensis
in ABCB6KD macrophages exposed to meglumine antimoniate.
A dose-dependent effect of the drug was observed in parasite
killing both in WT and knockdown macrophage cell lines
(Figure 6a). Intracellular parasite survival was significantly higher
in ABCB6KD cells, with an increase in parasite survival of 24% and
11% after exposure at drug concentrations of 16 and 32 mg/mL
Sb, respectively, in knockdown cells compared with WT (Figure 6a).
Enhanced survival of L. panamensis in ABCB6KD cells was not
the result of intrinsic permissiveness of these cells to infection or
Figure 2. Macrophage drug transporters and drug-metabolizing enzyme genes are modulated by L. panamensis (L.p) infection and exposure to
meglumine antimoniate (MA). (a) Heat map representation of the overall macrophage gene expression profile and (b) fold change values of drug
transporters and metabolizing enzymes of CL patients (eight responders and eight failures) induced by ex vivo infection with L. panamensis and
exposure to meglumine antimoniate (32 mg/mL Sb). Green indicates down-regulation and red indicates up-regulation. Clustering and correlation
analyses were performed using average linkage and uncentred correlation functions on Cluster3 software (a). Statistical differences were determined
by parametric or non-parametric analysis of variance tests, as appropriate, and significance established at P,0.05. Mean or median values are
represented according to distribution of data (b). This figure appears in colour in the online version of JAC and in black and white in the print version of JAC.
145
Gómez et al.
(a)
Infected
Uninfected
L.p-GFP
Merge
Staining
control
ABCB6
ABCB6
(b)
Figure 4. Subcellular localization of ABCB6 in uninfected and L. panamensis-infected macrophages. (a and b) ABCB6 is shown in red and intracellular
Leishmania in green in THP-1 macrophages infected with GFP-transfected L. panamensis (L.p-GFP). ABCB6 is detected at the cell membrane both in
infected and uninfected cells (white arrowheads) and surrounding the intracellular Leishmania (blue arrowheads). A Z-stack reconstitution of
sequential images taken at a Z scaling value of 0.38 mm is shown (a). Zoomed sequential Z-stack images show the specific localization of ABCB6 at the
periphery of the Leishmania-containing phagolysosome (b). This figure appears in colour in the online version of JAC and in black and white in the print
version of JAC.
intracellular parasite replication, as Leishmania survival in the
absence of drug was similar in WT and knockdown cells up to
96 h post-infection (Figure 6b). The antimicrobial effect of antimonials was not solely dependent on ABCB6 function; a trend of
increased parasite survival, although not statistically significant,
was also observed in both AQP-9KD (6%–19% increased survival)
and SLC7A11KD (10%–14% increased survival) cells compared
with WT macrophages (Figure S2, available as Supplementary
data at JAC Online).
Collectively, these findings support the function of ABCB6 as an
Sb transporter located both at the cell and phagolysosomal membranes and potentially modulating the exposure of intraphagosomal Leishmania to Sb-containing drugs.
Discussion
Investigations of Leishmania –macrophage interactions have
largely focused on the capacity of this parasite to modulate the
host immune response.23 Our findings expand the scope of host
cell functions modulated by Leishmania parasites and portray a
very dynamic interaction at the interface of infection and subsequent antimicrobial drug exposure.
In the present study, we have explored the role of human
macrophages in the antileishmanial effect of antimonials in
the context of clinical infections with Sb-susceptible Leishmania
146
strains. Upon exposure to meglumine antimoniate, the survival
of Sb-susceptible L. panamensis was significantly higher in macrophages from patients who failed treatment with antimonials, compared with those from patients who responded. This finding
substantiates the participation of host cell factors in the antimicrobial efficacy of antileishmanials and underscores the importance of
host cell determinants in optimizing therapeutic approaches to this
and other intracellular pathogens. Host cell transport and metabolism of antimicrobial compounds that target intracellular pathogens, and drug delivery to the specialized compartments where
these microbes reside and replicate, are crucial to efficacy. Previous
studies have addressed the importance of host cell drug transporters in infections caused by intracellular bacteria24,25 and viruses.26
We show that increased intracellular survival of Leishmania in
human macrophages after exposure to Sb correlates with low expression of the transporter ABCB6. Sb induced abcb6 gene expression in human macrophages, similar to the up-regulation induced
by the closely related metalloid As.18 shRNA gene knockdown of
ABCB6 revealed increased accumulation of Sb in ABCB6KD monocytes. This, together with the previously described As-transport
function of ABCB6,18 supports the function of ABCB6 as an Sb transporter. Biochemical analyses of the function of ABCB6 have shown
that this transporter acts as an efflux pump in the cell membrane
and as an importer when localized at intracellular membranes.17,19,21 This evidence, together with the phagolysosomal localization of ABCB6 in L. panamensis-infected macrophages, and
JAC
Leishmania and Sb modulate host drug response
(a) 100
300
Parasite survival (%)
Intracellular accumulation
of Sb (ng/106 cells)
(a)
200
100
0
10
20
30
Exposure dose (mg/mL) Sb
40
Intracellular accumulation
of Sb (ng/106 cells)
300
16 mg/mL
32 mg/mL
200
100
Parasite survival
(relative light units)
(b)
(b)
*
80
60
*
40
20
0
MA
(mg/mL Sb)
0
WT
ABCB6KD
0
8
32
16
6000
WT
ABCB6KD
4000
2000
0
2
0
0
24
48
Time of Sb exposure (h)
72
24
48
Time (h)
72
96
Figure 5. Characterization of intracellular uptake of Sb in THP-1 cells. (a)
Intracellular Sb content in WT THP-1 monocytes is linear over a range of
exposure to meglumine antimoniate (8– 32 mg/mL Sb) for 24 h. (b) Sb
accumulation curve constructed based on intracellular Sb concentrations
after exposure of WT THP-1 macrophages to meglumine antimoniate at
concentrations of 16 and 32 mg/mL Sb during a time course of 24 –72 h.
Graphs represent means+SEM of experiments performed in triplicate.
Figure 6. Knockdown of ABCB6 promotes intracellular survival of
L. panamensis upon exposure to meglumine antimoniate (MA). (a) Parasite
survival in WT and ABCB6KD macrophages infected for 24 h with L.p-LUC
001 and exposed to serially increasing concentrations of meglumine
antimoniate for an additional 24 h, expressed as percentage survival over
infected and untreated controls. (b) Parasite survival in WT and ABCB6KD over
a 96 h time course in the absence of meglumine antimoniate expressed
as absolute relative light units. Data are presented as means+SEM of
experiments performed in triplicate. Statistical significance was considered as
P,0.05 when applying unpaired t-test analysis.
the enhanced survival of Leishmania in ABCB6KD cells when
exposed to antimonials, substantiate its role as a regulator of
Sb-mediated intraphagosomal parasite killing. That the level of expression of ABCB6, a lysosomal/phagolysosomal drug transporter,
alters the antileishmanial effect of antimonials in infected human
macrophages highlights that the host cell-dependent regulation of
drug accumulation and activation within intracellular organelles
are important mechanisms of drug efficacy during the drugmediated parasite killing.27
The antileishmanial effect of antimonials does not uniquely
depend on ABCB6 expression/function; knockdown of macrophage
ABCB6 did not completely abrogate the capacity of antimonials to
kill intracellular Leishmania. Evaluation of the functional effect of
other host transporters, including AQP-9 and SLC7A11, which
were positively correlated to ABCB6 expression, showed a trend
of increased parasite survival in knockdown macrophages, suggesting a contribution of these transporters in the antimicrobial
effect of antimonials. It has been previously shown that the overexpression of AQP-9 in the K562 leukaemic cell line leads to intracellular accumulation of Sb and sensitizes these cells to AsIII and
SbIII.28 SLC7A11, a cystine/glutamate exchanger, plays an important role in the cellular redox balance through the maintenance of
reduced GSH levels;29,30 reduced GSH is essential in the
GSH-mediated activation of Sb (SbV SbIII).31 Hence, concomitant
repression of aqp-9, slc7a11 and abcb6 could promote the intracellular survival of Leishmania during exposure to antimonials by reducing AQP-9-mediated drug uptake, decreasing distribution to
the parasite-containing phagolysosome through ABCB6 and reducing conversion into bioactive SbIII indirectly by the function of
SLC7A11.
mt2a gene expression, similarly to abcb6 expression, is strongly
induced by As32,33 and MT2A-null mice have increased sensitivity to
As-induced toxicity.34,35 We found that mt2a expression was
strongly induced by Sb and that this increased mt2a expression
negatively correlated with intracellular parasite survival. Increased
levels of MT2A may promote intracellular Sb sequestration, thereby
reducing cellular toxicity, while promoting efficient Sb delivery to
the phagolysosome as MT2A is localized in the cytoplasm and lysosomal compartments.33 Although functional validation is necessary, and is the focus of our current research, the correlation of
increased mt2a gene expression with lower rates of intracellular
parasite survival within macrophages from CL patients supports
this hypothesis. Consistent with the multifactorial nature of the
therapeutic response, we have observed that patients with acute
147
Gómez et al.
disease who failed treatment with meglumine antimoniate had
significantly larger cutaneous lesions at the time of diagnosis
than patients who responded to treatment (Table 1). Larger
lesions are often accompanied by increased local immunopathology, which could contribute to a poor treatment outcome.
Among the host cell molecules identified in this study, macrophage alox5, aqp-9 and mt2a have previously been shown to be
modulated by infection with Leishmania donovani. 36 – 38 In addition,
monocytes from visceral leishmaniasis patients in India from a geographical area where L. donovani infections are frequently unresponsive to Sb, and who failed treatment with antimonials, were shown
to accumulate less intracellular Sb than cells from responding
patients. This difference was attributed to an induction of host cell
MRP-1 and P-gp expression as a result of infection with Sb-resistant
L. donovani strains, but not Sb-susceptible strains, leading to active
drug efflux.11 In agreement with these observations, in our study
of CL and treatment failure in the context of infections with
Sb-susceptible L. panamensis, we did not detect significant variation
in intracellular Sb or gene expression of mrp-1 and p-gp in macrophages from patients who failed or responded to meglumine
antimoniate. Together, these results portray the complexity of the
host cell mechanisms underlying the antileishmanial effect of antimonials and the potential impact of the infecting Leishmania
species and strain. Based on these findings, we suggest two nonexclusive host cell-dependent mechanisms operating to promote
the intracellular survival of Leishmania during antimonial drug
exposure: (i) the survival of Sb-resistant parasites could be attributed
to both intrinsic or acquired mechanisms of drug resistance coupled
with reduced intramacrophage accumulation of the drug through
up-regulation of host cell MRP-1 and P-gp; and (ii) the survival of
Sb-susceptible parasites could result from reduced exposure of the
intraphagosomal parasite to the drug through decreased expression
of host cell ABCB6.
Notwithstanding the individual function of the molecules validated in this study, the translation of these observations to therapeutic outcomes and the generalization of these data to infections
with other Leishmania species and clinical manifestations will
require consideration within the context of a multimarker profile
defining immune as well as pharmacological functions. These
issues are not confined to leishmaniasis; the influence of host–
pathogen –drug interactions on the pharmacological response
challenges the exclusive focus of antimicrobial strategies on the
targeting of the pathogen without consideration or exploitation
of host cell determinants of efficacy.
Acknowledgements
We gratefully acknowledge the patients who participated in this study, the
support of Dr David Gregory (Department of Environmental Health, Harvard
University) for training in gene silencing through shRNA, the CIDEIM BioBank
team for the culture, typing and evaluation of drug susceptibility of clinical
strains and Dr Neal Alexander (Epidemiology and Biostatistics Unit, CIDEIM)
for insightful discussions.
Funding
This work received financial support from COLCIENCIAS grant
# 222949326161 and the UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) Re-entry
grant # B00032. L. J. R. and D. A. V. are recipients of COLCIENCIAS Young
148
Investigator Awards, Contract #797-2009 and #0040-2012. V. M. B. and
M. A. G. were supported by Swiss National Science Foundation grant
#IZ70ZD_131421 and Fogarty/National Institutes of Health (NIH) training
grant D43 TW006589.
Transparency declarations
None to declare.
Supplementary data
Tables S1, S2 and S3, and Figures S1 and S2 are available as Supplementary
data at JAC Online (http://jac.oxfordjournals.org/).
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