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Acta Tropica
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Department of Parasitology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, Sichuan, PR China
West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu, Sichuan, PR China
c
Animal Disease Prevention and Food Safety Key Laboratory of Sichuan Province, Sichuan University, Chengdu, Sichuan, PR China
b
a r t i c l e
i n f o
Article history:
Received 29 March 2015
Received in revised form 27 August 2015
Accepted 12 October 2015
Available online 23 October 2015
Keywords:
Leishmania
Apoptosis
THP-1
RAW264.7
Cycloheximide
a b s t r a c t
Leishmania spp. are able to survive and proliferate inside mammals mononuclear phagocytes, causing
Leishmaniasis. Previous studies have noted that the regulation of apoptosis in host cells by these parasites
may contribute to their ability to evade the immune system. However, current results remain unclear
about whether the parasites can promote or delay the apoptotic process in host cells, because the regulatory effect of Leishmania was assumed to be strain-, species- and even infection time-dependent. The aim
of this study was to investigate whether the Sichuan isolates of Chinese Leishmania (SC10H2) can alter the
process of intrinsic apoptosis induced by cycloheximide in different types of macrophage cell lines and to
determine in which steps of the signaling pathway the parasites were involved. Human THP-1 and mouse
RAW264.7 macrophages were infected by SC10H2 promastigotes followed by cycloheximide stimulation
to assess the alteration of intrinsic apoptosis in these cells. The results indicated that SC10H2 infection
of human THP-1 macrophages could promote the initiation of intrinsic apoptosis, but completely opposite results were found in mouse RAW264.7 macrophages. Nevertheless, the expression of Bcl-2 and the
DNA fragmentation rates were not altered by SC10H2 infection in the cell lines used in the experiments.
This study suggests that SC10H2 promastigote infection is able to promote and delay the transduction
of early apoptotic signals induced by cycloheximide in THP-1 and RAW264.7 macrophages, revealing
that the regulation of intrinsic apoptosis in host cells by SC10H2 in vitro occurs in a host cell-dependent
manner. The data from this study might play a signicant role in further understanding the relationship
between Leishmania and different host cells.
2015 Elsevier B.V. All rights reserved.
1. Introduction
Protozoan parasites of the genus Leishmania, obligate intracellular pathogens, primarily invade macrophages as well as monocytes
in mammals and cause zoonotic leishmaniasis. Depending on the
different organs where the pathogen is found, human leishmaniasis
can be categorized into three main forms: visceral, cutaneous and
mucocutaneous leishmaniasis. Substantial human infection can be
caused by at least 21 of 30 species of the genus Leishmania in dif-
ferent regions (Chandra and Naik, 2008; Olivier et al., 2005). In the
relationship between Leishmania and humans, the sand y plays
an important role as a vector that carries and transmits the parasite to human beings. The two forms of Leishmania, promastigotes
and amastigotes, represent the different stages in sand ies and
humans, respectively. When transmitted by sand ies, promastigotes develop into amastigotes in macrophages and continue to
proliferate inside of the ies. An intriguing component of Leishmania pathogenesis is the ability to evade the immune system,
which promotes successful survival and further proliferation by
protecting the parasites from the killing effect of macrophages.
To avoid immune elimination and guarantee their survival
within hosts, Leishmania can evade and suppress the immune
defense system of hosts by developing multiple tactics, such
as modifying the complement system and phagocytosis process,
interfering with signaling pathways in macrophages, modulating
102
103
Table 1
Primers used for reverse transcription PCR.
Primer sequence (5 3 )
cDNA
Transcript
Sense
Antisense
Human
Bcl-2
cIAP1
XIAP
GAPDH
TTTGAGTTCGGTGGGGTCAT
CTCCAGCCTTTCTCCAAACCC
AGACACCATATACCCGAGGAACC
GAAGGTGAAGGTCGGAGTCA
TGACTTCACTTGTGGCCCAG
CCAGTTACTGAGCTTCCCACCAC
GTTTTCCACCACAACAAAAGCAC
TTCACACCCATGACGAACAT
Mouse
Bcl-2
c1AP-1
XIAP
-actin
GCACCCACTCCCTTCATACAAT
GTGGTTAAAGCAGCCTTGGA
TTGGAACATGGACATCCTCA
ATATCGCTGCGCTGGTCGTC
ACGCAGGTTACATTCGTCTTCC
CATTGGTGTCACACACGTCA
CGCCTTAGCTGCTCTTCAGT
AGGATGGCGTGAGGGAGAGC
Table 2
PCR cycles and temperatures for amplication of the different human cDNA.
Thermo-cycling conditions
Melting
Annealing
Extension
Final extension
Cycles
Bcl-2
cIAP1
XIAP
GAPDH
Temperature
Duration
Temperature
Duration
Temperature
Duration
Temperature
Duration
95 C
63 C
72 C
72 C
35
30 s
30 s
1 min
5 min
95 C
59 C
72 C
72 C
28
30 s
30 s
1 min
5 min
95 C
61 C
72 C
72 C
29
30 s
30 s
1 min
5 min
94 C
55 C
72 C
72 C
30
2 min
1 min
1 min
5 min
104
Table 3
PCR cycles, and temperatures for amplication of the different mouse cDNA.
Thermo-cycling conditions
Melting
Annealing
Extension
Final extension
Cycles
Bcl-2
cIAP1
-actin
XIAP
Temperature
Duration
Temperature
Duration
Temperature
Duration
Temperature
Duration
94 C
56 C
72 C
72 C
30
30 s
30 s
30 s
5 min
94 C
55 C
72 C
72 C
30
2 min
1 min
1 min
5 min
94 C
55 C
72 C
72 C
30
30 s
30 s
1 min
5 min
95 C
59 C
72 C
72 C
30
30 s
30 s
1 min
5 min
Fig. 1. The morphology and infectivity of Leishmania SC10H2. (A) Leishmania SC10H2 promastigotes visualized by Wrights Dye. With an infection ratio of 10:1
(parasitesmacrophages) and an infection time of 4 h, the culture medium with uninfected promastigotes was harvested and stained for agella, nuclei and kinetoplasts in
each individual under an optical microscope. The results are from one representative experiment out of three. (B) Leishmania SC10H2 amastigotes visualized by Wrights
Dye. With the same infection ratio and time as above, the infected cells on slides with transformed amastigotes in their cytoplasm could be observed by Wrights Dye. The
results are from one representative experiment out of three. (C) The transformed SC10H2 amastigotes within THP-1 macrophages visualized with a transmission electron
microscope. A single amastigote with the structure of both the cell nucleus and the kinetoplast could be found close to the nuclear membrane of the macrophage under the
transmission electron microscope. The results are from one representative experiment out of two.
105
Fig. 2. Loss of the mitochondrial transmembrane potential of macrophages shown by uorescence. With cycloheximide stimulation (5 g/ml for THP-1 cells and 2.5 g/ml
for RAW264.7 cells) for 16 h after a 4 h infection time (infection ratio 10:1), the infected THP-1 cells (A1) had a greater ratio of early apoptotic cells to normal cells than that of
control cells (A2), representing an increased loss of mitochondrial transmembrane potential in these cells. In contrast to the results found in THP-1 cells, infected RAW264.7
cells treated with cycloheximide demonstrated a decreased loss of mitochondrial transmembrane potential (A3) by having a greater ratio of normal cells to early apoptotic
cells compared with that of control cells (A4). (B1) and (B2) represent the infected and uninfected THP-1 cells without stimulation, respectively. (B3) and (B4) demonstrate
infected and uninfected RAW264.7 cells without stimulation, respectively. The orangered color indicates normal cells, while the green color indicates early apoptotic cells.
The results are from one representative experiment out of two. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of
this article.)
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Fig. 3. Caspase-3 and caspase-9 activities of human THP-1 macrophages and mouse RAW264.7 macrophages. In (A), CHX+ and CHX represent the caspase-3 activities
of infected and un-infected THP-1 cells for 4 h (10:1 ratio) followed by stimulation with cycloheximide (5 g/ml) for 16 h. + and Represent the caspase-3 activities of
infected and uninfected THP-1 cells for 4 h without stimulation of the inducer for 16 h. The indices of (C) represent the same experimental conditions as (A), with caspase-9
activity measured in place of caspase-3. In (B), CHX+ and CHX represent the caspase-3 activities of infected and uninfected RAW264.7 cells for 4 h followed by stimulation
of cycloheximide (2.5 g/ml) for 16 h. + and Represent the caspase-3 activities of infected and uninfected RAW264.7 cells for 4 h without cycloheximide stimulation for
16 h. The indices of (D) represent the same experimental conditions as (B), with the caspase-9 activity measured in place of caspase-3. The unit of caspase activity was
absorbance/(time in h concentration of protein). The results represent the mean SD of three independent experiments. * P 0.05, ** P 0.01 and *** P 0.001.
107
Fig. 4. Different expression levels in THP-1 macrophages and mouse RAW264.7 macrophages by Western blot and PCR. (+) indicates the XIAP, Bcl-2 and c-IAP1 expression
levels of macrophages stimulated with cycloheximide (5 g/ml for THP-1 cells and 2.5 g/ml for RAW264.7 cells) for 16 h after infection with Leishmania (10:1) for 4 h. ()
Indicates cells stimulated with cycloheximide for 16 h without parasites infection. GAPDH was the housekeeping control for THP-1 cells, and -actin was the housekeeping
control for RAW264.7 cells. The molecular weight of each antigen tested in the Western blot is: 53 kDa for XIAP, 28 kDa for Bcl-2, 37 kDa for GAPDH and 43 kDa for -actin.
The results are from one representative experiment out of three.
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Fig. 5. A TUNEL assay shows the DNA fragmentation level of THP-1 and RAW264.7 macrophages following apoptosis induction and at the baseline level. (A1) revealed that
the DNA fragmentation level of THP-1 cells infected with SC10H2 (infection ratio of 10 parasites:1 macrophage) for 4 h followed by stimulation with cycloheximide for 16 h
were similar to that of uninfected cells under the same conditions (A2). When treated with only parasites for 4 h without an apoptotic inducer, the uninfected THP-1 cells
(B2) and SC10H2-infected cells (B1) also had the same DNA fragmentation level. (C1) and (C2) demonstrate the positive and negative control, respectively.
(A3) shows that the DNA fragmentation level of RAW264.7 cells infected by SC10H2 (infection ratio of 10 parasites:1 macrophage) for 4 h followed by stimulation with
cycloheximide for 16 h were similar to that of uninfected cells under the same conditions (A4). When treated with parasites only for 4 h, the uninfected RAW264.7 cells (B4)
and SC10H2-infected cells (B3) also had the same DNA fragmentation level. (C3) and (C4) demonstrate the positive and negative control, respectively. The results are from
one representative experiment out of three.
109
et al., 2005). All of these results are based on different concentrations of the different chemical inducers or different time points
to measure DNA fragments. The inducers used for these experiments included actinomycin D, staurosporine, cycloheximide and
campothecin (Donovan et al., 2009; Akarid et al., 2004; Ruhland
et al., 2007; Lisi et al., 2005) in different concentrations. A dose gradient study of cycloheximide in rats suggests that below a certain
concentration, cycloheximide has no effect on the inhibition of protein synthesis (Alessenko et al., 1997). Nevertheless, it is unlikely
that the doses (5 g/ml for THP-1 cells, 2.5 g/ml for RAW264.7
cells) were below this threshold, because these concentrations
were appropriate according to other experiments for the induction of DNA fragmentation (Kim et al., 2000; Wang et al., 2005;
Cipriani et al., 2001). Another condition that should be considered
is the time points used for the measurements. Studies using chemical inducers primarily chose a time point of approximately 28 h in
total (with infection for 4 h and stimulation for 24 h) while our time
point was 20 h (with infection for 4 h and stimulation for 16 h). In
particular, Donovan et al. (2009) used the same inducer with the
same infection and stimulation time points as we did, but the concentration of cycloheximide was not stated. An interesting result
obtained from nding of induced apoptosis in THP-1 macrophages
infected by Leishmania was the absence of DNA fragmentation
(Nagata and Apoptotic, 2000), indicating that apoptosis could occur
independently of DNA fragmentation or nuclear degeneration. The
incomplete activation of macrophage apoptosis was also demonstrated in the investigation of L. pneumophila and host cell apoptosis
(Abu-Zant et al., 2005). However, the results of our study indicate
that even the control cells, which were treated with cycloheximide
alone, did not show increased DNA fragmentation rates, indicating
that the particular type of apoptosis induction mentioned above
was not the cause of the absence of DNA fragmentation. Therefore,
we attributed the absence of DNA fragmentation to the time points
that we chose for the experiments. Because the exposure time of
macrophages to cycloheximide was relatively short, the nal step
of apoptosis may not have occurred. Indeed, when we conducted
the experiment in THP-1 macrophages with prolonged time points
(24 h of infection followed by 24 h of stimulation with cycloheximide), we observed a difference in the DNA fragmentation rates
between infected and un-infected cells (data not shown), demonstrating that the absence of DNA fragmentation was due to the short
challenge time. Surprisingly, after 48 h, infected THP-1 cells displayed a decreased induction of apoptosis, which is the opposite of
the result that we found at the 20 h time point. Therefore, our data
suggest that Leishmania both induce and prevent apoptotic signal
transduction. In the early stage, they promote apoptosis to avoid
killing by inammatory factors, while in the late stage, they prevent the apoptosis to proliferate as much as possible inside host
cells.
5. Conclusions
In conclusion, the SC10H2 isolates of Chinese Leishmania are
able to infect macrophages in vitro. Cycloheximide-induced intrinsic apoptosis is regulated by these parasites, but has contrasting
outcomes in THP-1 and RAW264.7 cells. Therefore, it is likely that
Leishmania adapt to the particular environment of the individual host cells from different mammals and thus have different
regulatory effects. However, there was no difference in the DNA
fragmentation rates between the infected and un-infected cells
during apoptosis induction, despite the signicant differences in
the early signs of apoptosis, indicating that the time point chosen
is too early for the execution step. Future studies should focus on
the possibility of reversing of apoptosis at prolonged time points in
110
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (81171607, J1103604), the National Project
of Important Infectious Diseases of China (2008-ZX10004-011),
and the Foundation for Young Teachers in Sichuan University
(2012SCU11093).
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