Experimental Parasitology 122 (2009) 353–356

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Experimental Parasitology
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Research Brief

Leishmania (Viannia) peruviana (MHOM/PE/LCA08): Comparison of THP-1 cell

and murine macrophage susceptibility to axenic amastigotes
for the screening of leishmanicidal compounds
Germán González a, Denis Castillo a, Yannick Estevez b, Thomas Grentzinger c, Eric Deharo b,c,*
a
Laboratorios de Investigación y Desarrollo, Facultad de Ciencias y Filosofía, Universidad Peruana Cayetano Heredia (UPCH), Av. Honorio Delgado 430, SMP, Lima, Peru
b
Université de Toulouse, UPS, UMR 152 (Laboratoire de pharmacochimie des substances naturelles et pharmacophores redox), 118, rte de Narbonne, F-31062 Toulouse cedex 9, France
c
IRD, UMR-152, Mission IRD Casilla 18-1209 Lima, Peru

a r t i c l e i n f o a b s t r a c t

Article history: This study, undertaken to compare the susceptibility of THP-1 cells and murine peritoneal macrophages
Received 19 October 2008 to Leishmania peruviana amastigotes, obtained THP-1 infection with 10 parasites/cell compared to 2 par-
Received in revised form 6 May 2009 asites/murine macrophage. The parasite burden was maximal at 72 h post infection (h.p.i.) for THP-1
Accepted 8 May 2009
cells, while it was still increasing at 120 h.p.i. for murine macrophages. Since in both cases the infection
Available online 19 May 2009
with L. peruviana affected cell viability, we recommend evaluating any leishmanicidal activity at 72 h.p.i.
Amphotericin B reduced Leishmania infection by 50% at concentrations of 0.1 lM in THP-1 and murine
Keywords:
macrophages at 72 h.p.i.
THP-1
Murine macrophages
Our results demonstrate that amastigotes of L. peruviana can infect THP-1 cells and murine macro-
Leishmania phages and indicate the suitability of this model to screen compounds for leishmanicidal activity.
Leishmania peruviana Ó 2009 Elsevier Inc. All rights reserved.
Leishmanicidal
Amphotericin B

1. Introduction Hiwot et al., 1992) to compare the susceptibility of L. peruviana

amastigote in human acute monocytic leukemia cells (THP-1) and
Leishmaniasis, a parasitic disease caused by the genus Leish- murine macrophage infected cells.
mania, is transmitted to humans by infected female sandflies. More
than 12 million clinical cases are annually reported, with an infec- 2. Materials and methods
tion range that spans four continents (Desjeux, 2004). This disease
continues to be of global importance as treatments require paren- 2.1. Chemicals
teral administration, produce strong collateral effects, and are costly
for the majority of the populations in affected countries. In Peru, M199 medium was purchased from Invitrogen, L-glutamine,
cutaneous and mucocutaneous forms of leishmaniasis are endemic antibiotics and fetal bovine serum from BioWitaker Cambrex, and
in 74% of the country (Sánchez et al., 2004). Among the five species the remaining chemicals were obtained from Sigma–Aldrich.
presentLeishmania (Viannia) braziliensis is responsible for mucocuta-
neous lesions while Leishmania (Viannia) peruviana, Leishmania
2.2. Parasites
(Viannia) guyanensis, Leishmania (Viannia) lainsoni and Leishmania
(Leishmania) amazonensis produce cutaneous forms. For unclear
L. peruviana (MHOM/PE/LCA08) was maintained in the promas-
reasons, L. peruviana strain at times causes mucocutaneous signs
tigote stage in a biphasic medium (blood agar with 0.89% NaCl, pH
similar to those caused by L. braziliensis (Llanos–Cuentas, personal
7.4) at 24 °C, with sub-passage every 3–4 days. Promastigotes
communication). However, no previous research has provided a
(5  106 parasites) were then transferred to 25 cm2 tissue culture
tailored model to study leishmanicidal drugs against L. peruviana.
flasks containing 5 ml of M199 medium supplemented with 10%
Our research adapts a model (Ogunkolade et al., 1990 and Gebre-
fetal bovine serum (FBS), pH 7.4. After 4 days, exponential phase
promastigotes were centrifuged for 10 min at 1500g and 4 °C.
* Corresponding author. Address: Université de Toulouse, UPS, UMR 152 The supernatant was discarded and replaced by fresh M199 med-
(Laboratoire de pharmacochimie des substances naturelles et pharmacophores
ium supplemented with 20% FBS, pH 5.5. Axenic amastigotes trans-
redox), 118, rte de Narbonne, F-31062 Toulouse cedex 9, France. Fax: +51 1 441 32
23 22. formation was then induced by increasing the temperature to
E-mail address: ericdeharo@gmail.com (E. Deharo). 34 °C and incubating for 96 h (Teixeira et al., 2002).

0014-4894/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.exppara.2009.05.005
354 G. González et al. / Experimental Parasitology 122 (2009) 353–356

2.3. Human cells mean number of intracellular amastigotes was determined three
times in 100 cells.
THP-1 monocytic cells (ATCC TIB-202) were maintained in
25 cm2 tissue culture flasks with RPMI 1640 medium supplemented
2.5.3. Evaluation and comparison of drug activity in murine
with 5 mM L-glutamine, 100 U/ml penicillin, 100 lg/ml streptomy-
macrophages and THP-1 infected cells
cin, and 10% FBS at 37 °C and 5% CO2. Sub-passage occurred every
At 24 h.p.i., macrophages and THP-1 cells were infected, as de-
2 days, following Worley et al. (2003). Cells were centrifuged
scribed above, and were exposed to amphotericin B for the follow-
(10 min, 200g, 4 °C) and then diluted in fresh medium. The trypan
ing 96 h. Cells were then fixed with methanol, stained with
blue exclusion method was used to determine cell concentration
Giemsa, and examined at 1000 by light microscopy. The number
and viability. Cell concentration was adjusted to 2  105 living
of intracellular amastigotes was determined in 300 cells. Following
cells/ml; 4  4 mm sterile cover glasses and 50 ll of this dilution
Delorenzi et al. (2001) the percentage of infection rate (%IR) of each
were placed in each well of a 96-well plate (Castillo et al., 2007). Fifty
culture was calculated as follows:
microliter of fresh medium supplemented with 40 ng/ml final con-
%IR = 100 – (infection rate of the treated culture /infection rate
centration of phorbol-12-myristate-13-acetate (PMA) was added
of the untreated culture)  100. IC50 was also calculated as the
to each well and plates were incubated for 48 h at 37 °C, 5% CO2 to
dose capable of a 50% reduction in the number of infected cells
permit cell differentiation into macrophages (Park et al., 2007) and
(calculated using the Excel trend formula). All experiments were
adhesion to cover glasses. Plates were washed twice with pre-
performed in triplicate. ANOVA was used to test for statistical sig-
warmed RPMI 1640 to remove PMA, and 100 ll of fresh complete
nificance of differences (Epi-Info, Statview student program). The
RPMI 1640 medium were then added. Differentiated cells were
total parasite burden was calculated as a mean number of amastig-
maintained until experiments were conducted.
otes per cells X the number of infected macrophages (or THP-1).
2.4. Murine cells
2.5.4. Evaluation and comparison of viability of murine macrophages
and THP-1
Murine macrophages were harvested from peritoneal cavities of
Murine macrophages and THP-1 cells were deposited on sterile
6–8 week-old female BALB/c mice in ice-cold M199 medium sup-
cover glasses at the bottom of each well of a 96-well plate. Viability
plemented with 10% FBS (Sauvain et al., 1993). Extracted cells were
was determined by trypan blue dye exclusion method (Castillo
immediately deposited on sterile 4  4 mm cover glasses and
et al., 2007) until 120 h.p.i in infected (as described above) or
placed in each well of a 96-well plate. Plates were incubated for
non-infected cells.
24 h at 37 °C, 5% CO2 to allow cell adhesion (Castillo et al., 2007).
For each 4  4 mm cover glass, four 200 lm2 zones were ran-
Pre-warmed complete M199 medium was used twice to remove
domly selected. The number of viable cells detected at 24 h.p.i.,
non-adherent cells. A neutral red method (Fautz et al., 1991) was
was considered as 100%. The% of viable cells at 48, 72, 96 and
employed to determine cell concentration. Approximately
120 h were then calculated as follows:
7  104 viable cells were deposited in each well for adhesion.
ðNumber of viable cells at 48; 72; 96 or 120 h:p:i:=number of
2.5. Procedure viable cells at 24 h:p:i:Þ  100:

2.5.1. Cytotoxicity against murine peritoneal macrophages and THP-1 All experiments were performed in triplicate. Mann–Whitney
cells statistical test was used to compare 24 h.p.i. and the others (Epi-
Differentiated murine peritoneal macrophages and THP-1 cells Info, Statview student program).
were treated with amphotericin B and the trypan blue dye exclusion
method was used (Castillo et al., 2007). The prepared plates, as
described above, were used to test serial dilutions of amphotericin 3. Results and discussion
B. Dilutions of 10, 1 and 0.1 lM in complete medium were then
added to achieve a final volume of 100 ll. The culture was continued 3.1. Percentage of viable cells
for another 48 h. After this incubation, the number of viable cells was
scored by hematocytometer using 0.4% trypan blue solution in PBS. The % of viable non-infected cells (Fig. 1) was similar (no statis-
The half-maximal cytotoxic dose 50 (CC50) for each cell type was tical difference) in both cell types throughout the experiment
determined. All experiments were repeated three times.

2.5.2. Evolution of infection in murine peritoneal macrophages and

THP-1 cells
The medium of the 96-well plates with prepared cells, as de-
scribed above was replaced after 24 h (murine macrophages) or
48 h (THP-1) by L. peruviana amastigotes in M199 medium supple-
mented with 10% FBS, using an infection ratio of two amastigotes
per adhered murine macrophage and 10 amastigotes per adhered
THP-1 cell. The plates were incubated for 2 h at 32 °C, 5% CO2
and then washed three times with pre-warmed M199/RPMI1640
medium without FBS to remove free parasites. At this time (0 h),
percentage of infected macrophages was recorded to provide an
initial measure of infection. Subsequently, 100 ll of complete Fig. 1. Evolution of the percentage of viable L. (V.) peruviana infected murine
M199/RPMI1640 medium was added to each well, plates were re- peritoneal macrophages (grey bars) and THP-1 cells (white bars) determined from
turned to incubation, and parasitaemia was measured at 0, 24, 48, 24 to 120 h.p.i. (: statistically significant difference compared to 24 h.p.i, p < 0.05).
The number of viable cells detected at 24 h.p.i., was considered as 100%. The % of
72, 96 and 120 h post infection (h.p.i.). viable cells at 48, 72, 96 and 120 h were: (Number of viable cells at 48, 72, 96 or
Adherent cells were then fixed on slides with methanol, stained 120 h.p.i./number of viable cells at 24 h.p.i.)  100. All experiments were performed
with Giemsa, and examined at 1000 by light microscopy. The in triplicate.
 G. González et al. / Experimental Parasitology 122 (2009) 353–356
Fig. 2. Evolution of the total L. (V.) peruviana burden in peritoneal macrophages (grey bars) and THP-1 cells (white bars) at the indicated time post-infection. (: statistically
significant difference between both types of cells, p < 0.05).
(120 h). Interestingly, the viability of infected THP-1 cells de-
creased at 72 h, while 100% of infected murine macrophages were
still alive at that time. Then, at 96 and 120 h.p.i., in both cell types
the proportion of viable cells were statistically different (p < 0.05)
compared to the beginning of the experiment (24 h.p.i.).
3.2. Infection ratio
Murine peritoneal macrophages were infected with axenic
amastigotes of L. peruviana, with a ratio of 2 parasites/1 cell, while
in the case of THP-1 cells, it was necessary to start with a parasite
ratio five times higher (10 parasites/1 cell) to obtain a stable infec-
tion. Ogunkolade et al. (1990) used a similar infection ratio to in-
fect THP-1 cells with promastigotes (instead of amastigotes) of
old and new world Leishmania parasites (L. peruviana was not
tested in their experiment).
3.3. Parasite burden
The parasite burden (Fig. 2) increased from 0 to 120 h. for mur-
ine macrophage, while in the case of THP-1 cells, after 72 h, the
parasite burden decreased at 96 h.p.i. and increased again at
120 h.p.i. Differences (p < 0.05) appeared at 72 h.p.i. and 120 h.p.i.
between both types of cells.
3.4. Amphotericin B
Earlier literature (Mehta et al., 1984) reports that an amphoter-
icin B dose higher than 5 lM could alter murine macrophage mor-
phology without damaging the macrophage membrane. We found
(Table 1) that a dose of 5.8 ± 0.4 lM killed 50% of murine macro-
Table 1
Toxicity (CC50 on non-infected cells) and leishmanicidal activity (IC50 on infected
cells) of amphotericin B on murine peritoneal macrophages and THP-1 cells.
Type of cell CC50 IC50 72 h IC50 96 h IC50 120 h
Murine Mø 5.8 ± 0.4 0.1 ± 0.01 0.02 ± 0.01 0.03 ± 0.01
THP-1 7 ± 0.9 0.1 ± 0.01 0.08 ± 0.01 0.05 ± 0.04
Mø, macrophages; CC50, dose (lM) which kills 50% of non-infected cells; IC50, dose
(lM) which inhibits 50% of parasite growth.
355
phages, while in THP-1 cells, the same effect was produced with
7 ± 0.9 lM. This finding conforms to previous studies (Rogers
et al., 2003 and Sau et al., 2003), which showed that concentrations
>5 lM affect the viability of THP-1 cells.
The IC50 of amphotericin B was 0.1 lM at 72 h on both, murine
macrophages and THP-1 cells. Then, the IC50 decreased around five
times for murine macrophages and to a lesser extend for THP-1
cells. Buckner and Wilson (2005) and Escobar et al. (2002) also
found that amphotericin B inhibited 50% of L. amazonensis and
Leishmania mexicana growth in murine macrophages between 0.1
and 0.2 lM.
4. Conclusion
From the data obtained in our study, we conclude that L. peru-
viana amastigotes can infect both THP-1 cells and murine macro-
phages. According to our results, the best time to evaluate the
activity of leishmanicidal compounds in both cell types is
72 h.p.i. because:
– the parasite burden is maximal for THP-1 cells (probably
responsible of the decrease of their viability after 72 h.p.i.),
– the proportion of viable infected macrophages decreased after
72 h.p.i. (although the parasite burden still increased).
For the first time the IC50 of amphotericin B was shown to be
0.1 lM at 72 h.p.i.
L. peruviana amastigotes are thus suitable for the screening of
leishmanicidal compounds in both cell types, using amphotericin
B as positive control.
Acknowledgments
The authors thank Professor Jorge Arévalo of the Instituto de
Medicina Tropical ‘‘Alexander von Humboldt” of the Universidad
Peruana Cayetano Heredia, for providing THP-1 cells and L. peruvi-
ana LCA 08 strain. The authors gratefully acknowledge the financial
assistance of the ‘‘Oficina de Cooperación de la Embajada de Bélg-
ica” for funding the studies of Denis Castillo and German Gonzalez.
We also thank Ms. Marie-Jean Manrique and Julie Pontarollo for
the critical reading of this manuscript.
356 G. González et al. / Experimental Parasitology 122 (2009) 353–356

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