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Originally published online as doi:10.1189/jlb.1001877 on April 1, 2004

Published online before print April 1, 2004
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(Journal of Leukocyte Biology. 2004;76:95-103.)
© 2004 by Society for Leukocyte Biology

Leishmania major-mediated prevention of programmed cell death induction in infected macrophages is associated with the repression of mitochondrial release of cytochrome c

Khadija Akarid*,{dagger},1, Damien Arnoult*, Juliette Micic-Polianski*, Jamila Sif{dagger}, Jérôme Estaquier* and Jean Claude Ameisen*,2

* EMIU9922, INSERM/Université Paris 7, IFR02, APHP, Faculté de Médecine Xavier Bichat, France; and
{dagger} Département de Biologie, Faculté des Sciences, El Jadida, Morocco

2Correspondence: EMI-U 9922, INSERM/Université Paris 7, Faculté de Médecine Xavier Bichat, 16, Rue Henri Huchard, 75870 Paris Cedex 18, France. E-mail: ameisen{at}wanadoo.fr


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ABSTRACT
 
Leishmania are obligate, intracellular parasites of macrophages in their vertebrate hosts, including humans, in which they cause disease. Here, we report that in vitro infection with Leishmania major protects murine bone marrow-derived macrophages against programmed cell death (PCD) induced by deprival of macrophage-colony stimulating factor and delays PCD caused by treatment with staurosporine, a broad inducer of PCD. This preventive effect was observed in macrophages from L. major-susceptible BALB/c and L. major-resistant C57BL/6 mice, indicating that repression of PCD did not depend on genetic background-specific regulation of T helper cell type 1 (Th1)/Th2 cytokine secretion. Prevention of effector caspase activation and PCD was associated with a repression of mitochondrial release of cytochrome c and did not involve the nuclear factor-{kappa}B pathway. The capacity of L. major to delay PCD induction in the infected macrophages may have implications for Leishmania pathogenesis by favoring the invasion of its host and the persistence of the parasite in the infected cells.

Key Words: apoptosis • mononuclear phagocyte • intracellular parasite • mitochondria • caspase activation


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INTRODUCTION
 
Programmed cell death (PCD) is a genetically regulated process of cell suicide, whose most frequent phenotype is apoptosis, and is central to embryonic development, adult tissue homeostasis, and elimination of damaged cells [1 2 3 4 ]. PCD also plays an important role in the regulation of the immune response and more generally, in defense against infections [5 6 7 ]. In plants, insects, and mammals, the rapid induction of PCD in response to pathogen entry represents an evolutionarily conserved, protective response against infections [4 , 5 , 7 8 9 10 11 ].

In mammals (humans and mice), numerous intracellular pathogens have evolved various strategies involving a modulation of PCD that favors pathogen survival. One type of strategy, which plays an important role in the life-cycle of a wide range of viruses [12 13 14 15 ] and has also been described in some bacteria [16 17 18 19 20 ] and some intracellular parasites [21 22 23 24 25 ], is the repression of PCD in the host cells in which they replicate and/or persist. The second, converse type of strategy, described in some viruses [13 , 26 , 27 ], several bacteria species [28 29 30 31 32 ], and at least one intracellular parasite [33 ], involves the induction of PCD in immune effector cells, such as T lymphocytes or macrophages, which may target the infected cell for destruction.

Leishmania species are kinetoplastid protozoan parasites that colonize invertebrates and vertebrates and are obligate, intracellular parasites of macrophages in their vertebrate hosts, including humans, in which they cause disease [34 ]. As macrophages are the obligate host cell required for Leishmania persistence and propagation and one of the main effector cells allowing Leishmania killing [35 ], the potential outcome of Leishmania infection on macrophage survival—induction or repression of PCD—may have important implications for the understanding of the parasite pathogenesis. It has been reported previously that Leishmania donovani infection of murine bone marrow-derived macrophages (BMDM) represses macrophage PCD induced in vitro by deprival in macrophage-colony stimulating factor (M-CSF) through a mechanism involving the secretion of cytokines, in particular, tumor necrosis factor {alpha} (TNF-{alpha}), by the infected macrophages [22 ]. This previous study, suggesting that Leishmania-mediated enhancement of infected macrophage survival may be a virulence factor of the parasite, raises, however, several problems of interpretation. In particular, the sole criterion used to assess PCD prevention was oligonucleosomal DNA fragmentation, revealed by gel electrophoresis [22 ]. Several subsequent reports have shown that oligonucleosomal DNA fragmentation is dispensable for PCD induction and therefore, that the prevention of this apoptotic feature does not obligatorily correlate with the prevention of PCD [36 ]. In addition, this preventive effect of L. donovani infection on macrophage DNA fragmentation was only investigated in BMDM from BALB/c mice, which are unable to resist Leishmania infection in vivo [34 , 37 ]. Indeed, BALB/c mice respond to Leishmania by developing a T helper cell type 2 (Th2) cytokine response, resulting in a lack of immune control of parasite dissemination and in the development of lethal disease [34 , 37 ]. Mice with different genetic backgrounds, such as C57BL/6 mice, respond to Leishmania by developing a Th1 cytokine response, which allows immune control of parasite dissemination and recovery from disease [34 , 37 ]. Therefore, the absence of information about Leishmania effect on PCD in BMDM from resistant mice does not allow to discriminate between the possibility that Leishmania-mediated repression of macrophage PCD is an intrinsic virulence factor of the parasite, which does not depend on the genetic background of the host, and the possibility that it represents a host-dependent susceptibility factor to the parasite, related to the particular genetic control of cytokine secretion that renders the BALB/c mice unable to resist infection.

Here, we present findings that confirm and extend this previous observation [22 ] by showing that BMDM infection with another Leishmania species, Leishmania major, protects macrophages against PCD, induced not only by M-CSF deprival but also by treatment with the broad protein kinase inhibitor staurosporin, which causes PCD in all nucleated mammalian cells [38 ]. L. major infection prevented the typical features of apoptosis, including nuclear chromatin condensation, nuclear DNA loss, mitochondrial transmembrane potential ({Delta}{psi}m) loss, mitochondrial release of cytochrome c in the cytosol, and activation of the effector caspase-3. More importantly, L. major infection not only prevented the induction of these apoptotic features but also allowed macrophage survival. This preventive effect was observed in BMDM from the Leishmania-susceptible BALB/c mice and the Leishmania-resistant C57BL/6 mice, therefore implying that L. major-mediated prevention of BMDM PCD does not depend on genetic background- specific regulation of Th1/Th2 cytokine secretion. Finally, our findings strongly suggest that L. major does not exert its effect at the level of TNF-{alpha} or the related CD95 ligand signaling but at the level of mitochondria through the repression of the mitochondrial outermembrane permeabilization process, which allows the release of cytochrome c into the cytosol. Together, our findings imply that enhancement of infected macrophage survival is an intrinsic property of the Leishmania parasite, which may play a role in its life-cycle and is achieved through a control exerted at the level of its host cell mitochondria.


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MATERIALS AND METHODS
 
BMDM
To generate BMDM, bone marrow cells were cultured for 7 days in the presence of CSF-1 (M-CSF). Briefly, bone marrow cells were obtained by flushing the marrow from mouse femurs with RPMI 1640 and then dispersed, washed, and resuspended in RPMI 1640 (Gibco, Courbevoie, France), supplemented with 10% L-929 cell-conditioned medium as a source of M-CSF, 10% heat-inactivated fetal calf serum (FCS; Boehringer Mannheim, Meylan, France), 100 U penicillin (Gibco, Cergy-Pontoise, France) per ml, 100 mg streptomycin (Gibco, Courbevoie, France) per ml, and 2 mM L-glutamine. The cells were cultured for 7 days in 10 ml plastic petri dishes (Becton Dickinson, Pont De Claix, France), and confluent cells were cultured for an additional 16–20 h in complete RPMI 1640 without the M-CSF factor in the absence or presence of L. major. Cells were then incubated for up to 4 days (as indicated) in medium in the absence or presence of M-CSF (as indicated). BMDM cells used in this study were isolated from wild-type BALB/c and C57BL/6 mice (IFFA-CREDO, Saint Germain-sur-l’Abresle, France) or from CD95L-defective gld mice, from CD95-defective lpr mice, or from TNF-{alpha}–/–/lymphotoxin (LT){alpha}–/–mice, all with a C57BL/6 background (CNRS, Orleans, France).

Culture and infection of BMDM with L. major
L. major (World Health Organization strain WHOM/IR/-/173), a gift from Dr. Y. Jean-Francois. Garin (Saint Louis Hospital, Paris), were cultured at 27°C as promastigotes in M-199 (Gibco, Cergy-Pontoise, France) with 20% FCS, L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 mg/ml). Promastigotes were harvested from stationary-phase cultures, centrifuged, and added to quiescent BMDM at a ratio of 5/1, 10/1, or 40/1 parasites per BMDM for 15–18 h, after which time, noningested promastigotes were washed away, and infected BMDM were incubated in medium in the absence or presence of M-CSF. After 1 or 4 days (as indicated), adherent and nonadherent BMDM were harvested in the following manner. Nonadherent cells were collected from supernatants, and adherent BMDM were washed in 2 mg/ml glucose–phosphate-buffered saline (PBS) without calcium and magnesium, causing detachment of adherent cells. Adherent and nonadherent BMDM were collected, centrifuged, counted, and analyzed. Numbers of infected BMDM and of parasites per infected BMDM were assessed 48 h postinfection by Giemsa staining (Sigma, St. Quentin, France) [39 ].

In some experiments, BMDM were incubated with medium containing the nuclear factor (NF)-{kappa}B inhibitors MG-132 (Sigma) or sesquiterpene lactone parthenolide (PL; gift from Nicole Israel, Pasteur Institute, Paris) for 1 h before infection.

To assess the potential effect of TNF-{alpha} on BMDM survival, infected and uninfected BMDM were incubated with mouse recombinant TNF-{alpha} (1 ng or 100 ng/ml; R&D Systems, Abingdon, UK) during 24 h in the absence of M-CSF [22 ].

Evaluation of cell death and apoptotic features
Absolute numbers of living cells were assessed by counting adherent- and nonadherent-collected BMDM and excluding trypan blue-stained cells. Flow cytometric analysis was performed to assess {Delta}{psi}m and nuclear DNA loss. To evaluate changes in {Delta}{psi}m, cells were stained for 20 min at 37°C with 40 nmol/l potential, sensitive fluorescent dye 3.3'-diethyloxacarbocyanine (DiOC6; Molecular Probes, Eugene, OR). To evaluate nuclear DNA loss, cells were incubated with the nuclear dye acridine orange (0.1 µg/ml, Molecular Probes) for 10 min, as described previously [40 ]. Macrophages were gated under forward- and side-scatter parameters—apoptotic cells showing a characteristic, distinct peak of reduced fluorescence and forward-scatter below the peak of living cells.

Fluorescent microscopy analysis of nuclear chromatin condensation was performed by using Hoechst dye 33342 staining (1 µg/ml, Sigma) at room temperature, and fluorescent microscopy analysis of {Delta}{psi}m was performed by using DiOC6.

Immunostaining of cytochrome c
BMDM were cultured in duplicates in 24-well plates (Becton Dickinson) with round cover-slides (CML, France). Macrophages infected or uninfected by L. major were washed three times in PBS followed by fixation in freshly prepared 3% paraformaldehyde in PBS for 10 min. The fixed BMDM were washed three times in PBS for 15 min, each followed by permeabilization in 0.15% Triton X-100 in PBS for 15 min. The cells were then blocked for 60 min in blocking buffer (2% bovine serum albumin in PBS) followed by a 4-h incubation with a mouse monoclonal antibody against cytochrome c (1:200; 6H2B4, PharMingen, San Diego, CA). The cells were washed three times at 10 min each in blocking buffer followed by 1 h incubation with Texas Red-labeled goat anti-mouse immunoglobulin G (IgG; 1:500, Sigma).

Western blotting analysis of caspase-3
Cells were incubated in sodium dodecyl sulfate lysis buffer, boiled for 10 min, and centrifuged for 15 min at room temperature. Protein determination was done using DC protein assay (Bio-Rad Laboratories, Marnes La Coquette, France). Equal amounts of proteins were boiled for 5 min in 2x Laemmli sample and run on acrylamide gel and then transferred to polyvinylidene difluoride membrane (BioRad) and immunoblotted with a rabbit polyclonal anti-caspase-3 (a generous gift of Dr. Peter Vandenabeele, Ghent, Belgium) [41 ] and a mouse IgG2b antitubulin (KMX-1, Boehringer Mannheim). Western blots were then visualized using horseradish peroxidase-conjugated secondary antibodies (Amersham, Orsay, France), followed by enhanced chemiluminescence (Amersham).

Statistical analysis
Statistically significant differences were determined using the Student’s t-test.


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RESULTS
 
L. major-infected macrophages undergo delayed PCD in response to survival factor (M-CSF) deprival
To survive, all cell types depend on the constant repression of PCD induction by survival factors provided by other cells [2 , 3 , 42 ]; accordingly, cells cultured in vitro undergo apoptosis when deprived of survival factors [2 , 3 , 42 ]. Murine BMDM require the presence of M-CSF to survive in vitro and undergo apoptosis upon withdrawal of M-CSF from the culture. Removal of M-CSF from cultures of BMDM from wild-type C57BL/6 mice induced extensive cell death and cell loss, and only approximately 66% surviving cells remain in the culture after 1 day, and approximately 49% survive cells after 4 days (Fig. 1A ). The cells remaining in the culture after 1 day showed typical features of apoptosis, including nuclear chromatin condensation, assessed by fluorescence microscopy using the Hoechst dye 33342 (Fig. 1B) , nuclear DNA loss, assessed by flow cytometry analysis using the acridine orange nuclear dye (Fig. 1C) , and loss of {Delta}{psi}m, assessed by fluorescence microscopy (Fig. 1D) and flow cytometry (Fig. 1E) using the lipophilic fluorochrome DiOC6. When BMDM were infected in vitro by L. major promastigotes (at a ratio of 10 parasites per BMDM) for 24 h before M-CSF withdrawal, a preventive effect was observed on cell loss with 93% surviving cells 1 day after infection versus 66% after 1 day in the absence of infection and 82% 4 days after infection versus 49% after 4 days in the absence of infection (Fig. 1A) . The percentages of remaining cells with nuclear and mitochondrial features of apoptosis were also reduced (Fig. 1B 1C 1D 1E) . L. major infection at a ratio of five promastigotes per BMDM induced no significant cell death prevention (data not shown).



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  		Figure 1. Preventive effect of L. major infection on macrophage death induced by M-CSF deprival. (A) BMDM from C57BL/6 mice were not infected (No infection) or infected with L. major at a ratio of 10 parasites per BMDM (L. major infection 10/1), washed, and incubated for 4 days in the absence of M-CSF. Absolute numbers of living (trypan blue-excluding), remaining BMDM were assessed at days 1 and 4. After 24 h culture in the absence or presence of M-CSF, uninfected and L. major-infected BMDM were examined under a fluorescence microscope after staining with the Hoechst dye 33342 (B) to assess nuclear chromatin condensation and fragmentation or with the DiOC6 dye (D) to assess {Delta}{psi}m loss or were analyzed by flow cytometry after staining with the acridine orange dye (C) to assess DNA content or with DiOC6 (E) to assess {Delta}{psi}m loss. (B–E, a) Uninfected BMDM in the presence of M-CSF; (b) uninfected BMDM in the absence of M-CSF; (c) L. major-infected BMDM in the absence of M-CSF. Results are from one representative experiment out of six.

Incubation of BMDM from C57BL/6 mice with L. major promastigotes at this ratio of 10 parasites per BMDM led to a moderate level of infection with approximately 23% infected cells and approximately three parasites per infected cell (Fig. 2A ). In contrast, incubation of BMDM with L. major at a ratio of 40 parasites per BMDM led to extensive infection, with more than 70% infected cells and more than 12 parasites per infected cell (Fig. 2A) . However, the magnitude of the preventive effect on BMDM death induced by M-CSF deprival, as appreciated by {Delta}{psi}m measurements, was similar for infection rates of 10 parasites per BMDM and of 40 parasites per BMDM (Fig. 2B) . These finding suggested that the preventive effect of L. major infection on BMDM death may involve an indirect mechanism, such as the release of a survival factor by the infected macrophages. Alternately, it is possible that the important numbers of intracellular parasite per cell that follow infection at a ratio of 40 parasites per cell induce, by themselves, a toxic process that may counterbalance any potential survival advantage provided by this increased level of infection.



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  		Figure 2. Effect of the parasite per macrophage ratio and of the murine strain origin of macrophages on L. major-mediated prevention of macrophage death. (A) BMDM from C57BL/6 mice were infected with L. major at a ratio of 10/1 or 40/1 parasites per BMDM, washed, and incubated for 24 h. Percentages of infected cells in each condition were determined by counting 100 cells. The numbers of intracellular parasites per infected cell were determined following fixation and Giemsa staining by counting 100 cells. Results represent the mean ± SD of one representative experiment out of two performed in duplicate. (B) BMDM from C57BL/6 mice and from BALB/c mice were not infected (No infection) or infected (L. major infection) with L. major at a ratio of 10/1 or 40/1 parasites per BMDM, washed, and incubated for 24 h in the absence (M-CSF deprival) or presence (+M-CSF) of M-CSF. BMDM were then analyzed by flow cytometry using DiOC6 to assess {Delta}{psi}m. (C) BMDM from C57BL/6 and from BALB/c mice were not infected or infected with L. major at a ratio of 10 parasites per BMDM, washed, and incubated for 24 h in the absence or presence of M-CSF as in (B) and then analyzed by flow cytometry using acridine orange to assess DNA content. (B and C) Results represent mean ± SD of four independent experiments. *, P ≤ 0.05, and **, P ≤ 0.01.

The BMDM used in the above-mentioned experiments were from C57BL/6 mice, a murine strain that controls L. major infection and disease development in vivo, in particular, as a result of the capacity of their macrophages and other proinflammatory cells to secrete interleukin (IL)-12, a Th1-inducing cytokine in response to the parasite [34 , 37 , 43 ]. In contrast, other mice strains, such as BALB/c, fail to control L. major infection in vivo and develop lethal disease as a result of their failure to secrete IL-12 and to an early secretion of the Th2 cytokines IL-10 and IL-4 [34 , 37 , 43 ].

A previous study had reported that another Leishmania species, L. donovani, prevents oligonucleosomal DNA fragmentation induced by M-CSF deprival in BMDM from BALB/c mice through a mechanism involving cytokine secretion by the infected macrophages [22 ]. For this reason and as we previously observed that monocyte/macrophage survival is modulated by Th1 and Th2 cytokines [44 ], we investigated whether L. major infection may also repress death in BMDM from BALB/c mice, as it did in BMDM from C57BL/6 mice. {Delta}{psi}m analysis (Fig. 2B) and nuclear DNA content analysis (Fig. 2C) indicated that L. major infection protected BMDM from the Leishmania-resistant (C57BL/6) and -susceptible (BALB/c) mice strains against cell death induced by M-CSF deprival, suggesting that this preventive effect did not depend on the particular genetic control of Th1/Th2 cytokine secretion in response to Leishmania, which characterizes these two mice strains.

As infection of BMDM at the ratio of 10 parasites per cell, which caused only moderate levels of BMDM infection, provided a protection against cell death, which was as effective as that provided by infection at the ratio of 40 parasites per cell (Fig. 2B) , we decided to use this 10-parasites-per-cell ratio in all of the following experiments.

Apoptosis prevention induced by L. major infection does not involve modulation of TNF-{alpha} or CD95 death receptor-mediated signaling
Consistent with previous findings that TNF-{alpha} can act as a survival factor for monocytes [45 ], it has been suggested that the secretion of TNF-{alpha} by infected macrophages may account for the preventive effect of L. donovani infection on oligonucleosomal DNA fragmentation in BMDM from BALB/c mice in response to M-CSF deprival [22 ]. More generally, TNF-{alpha} and Fas ligand (CD95L) are two members of the TNF ligand family, which upon engagement of their TNF receptor (TNFR) and CD95 receptor, can induce in several cell types apoptosis or differentiation and/or proliferation, depending on the activation state of the cell bearing the receptors and on the nature of additional signals present in the environment [46 47 48 49 ]. Therefore, to investigate the potential role of a modulation of TNFR and CD95 signaling in the preventive effect of L. major on BMDM death, we used BMDM from TNF-{alpha}–/–/LT{alpha}–/– mice, from CD95L-defective gld mice, or from CD95-defective lpr mice (all of C57BL/6 background). As shown in Figure 3A , infection of BMDM with L. major led to a similar preventive effect on death induced by M-CSF deprival in the BMDM from TNF-{alpha}–/–/LT{alpha}–/–, gld, lpr, and wild-type mice. Thus, ours results strongly suggested that neither TNF-{alpha} receptor engagement nor CD95 engagement played any significant role in BMDM death induced by M-CSF deprival and in the preventive effect exerted by L. major infection.



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  		Figure 3. L. major-mediated prevention of macrophage death induced by M-CSF deprival does not involve a modulation of TNF-{alpha} or CD95 ligand-mediated death signaling. (A) BMDM from wild-type C57BL/6 mice or TNF-{alpha}–/–/LT{alpha}–/– mice, Fas (CD95)-defective lpr mice (LPR), and Fas ligand (CD95L)-defective gld mice (GLD), all of C57BL/6 background, were not infected (No infection) or infected with L. major at a ratio of 10 parasites per BMDM (L. major infection 10/1), washed, and incubated for 24 h in the absence (M-CSF deprival) or presence (+M-CSF) of M-CSF. BMDM were then analyzed by flow cytometry using DiOC6 to assess {Delta}{psi}m loss as described in Materials and Methods. Results represent mean ± SD of three independent experiments. *, P ≤ 0.05, and **, P ≤ 0.01. (B) BMDM from C57BL/6 wild-type mice, uninfected or infected with L. major at a ratio of 10 parasites per BMDM, were cultured for 24 h in the absence or presence of M-CSF, in the absence (–) or presence of murine TNF-{alpha} added in the culture at a concentration of 1 or 100 ng/ml. BMDM were then analyzed by flow cytometry using DiOC6 to assess {Delta}{psi}m loss. Results are from one representative experiment out of three.

Two additional experimental results, obtained with BMDM from wild-type C57BL/6 mice, further ruled out an involvement of TNF-{alpha} in the preventive effect exerted by L. major infection on macrophage death. First, the addition of a neutralizing antibody to murine TNF-{alpha} did not modify L. major-mediated repression of BMDM death in response to M-CSF deprival (data not shown). Second, when M-CSF deprival was combined with addition of exogenous murine recombinant TNF-{alpha} at low doses (1 ng/ml) or high doses (100 ng/ml), TNF-{alpha} showed no effect on the survival of uninfected and infected BMDM (Fig. 3B) .

L. major infection prevents mitochondrial release of cytochrome c and caspase-3 activation
With the possible exception of the engagement of the cell-surface death receptors of the TNFR family that directly induces caspase-8 activation [1 , 50 ], the activation of effector caspases, such as caspase-3, by most if not all proapoptotic stimuli requires a mitochondrial-dependent step that involves mitochondrial outer membrane permeabilization and leads to the release in the cytosol of mitochondrial intermembrane space proteins, including cytochrome c, which activates the caspase cascade [51 52 53 54 ].

We investigated whether L. major infection may exert its preventive effect on PCD at the level of mitochondria. As M-CSF deprival induced a rather slow and progressive death process, we also used staurosporine (0.5 µM) treatment to cause rapid (2 h) and synchronous BMDM death. Staurosporine is a broad protein kinase inhibitor that induces apoptosis in all human- and murine-nucleated cells studied so far [38 ], and L. major infection delayed BMDM death for at least 4 h in response to staurosporine (data not shown).

Immunofluorescence labeling of cytochrome c in intact cells, using fluorescence microscopy and an anticytochrome c antibody, showed a diffuse cytoplasmic staining in M-CSF-deprived and -uninfected BMDM and a granular staining in M-CSF-deprived, L. major-infected BMDM (Fig. 4 ), suggesting a reduction of cytochrome c release in the cytoplasm in the latter case. Staurosporine (0.5 µM) treatment for 2 h provided a confirmation, by showing a clear, diffuse cytoplasmic staining pattern in uninfected BMDM, and L. major-infected staurosporine-treated BMDM displayed a granular-staining pattern, implying a lack of cytosolic release of cytochrome c (Fig. 4) . Together, our findings indicated that the preventive effect of L. major infection on BMDM death is associated with a repression of mitochondrial outer membrane permeabilization ({Delta}{psi}m; Figs. 1E , 2B , and 3A ) and cytochrome c release in the cytosol (Fig. 4) . Mitochondrial release of cytochrome c in the cytosol induces the activation of the apoptosome, resulting in caspase-9 activation, and in the subsequent activation of effector caspases, such as caspase-3 [52 , 55 ]. As shown in Figure 5 , staurosporine treatment induced caspase-3 processing with a decrease of the inactive proform (32 kDa) and an appearance of the 21-kDa-active fragment [56 ] in uninfected BMDM but not in L. major-infected BMDM.



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  		Figure 4. L. major infection prevents mitochondrial release of cytochrome c in M-CSF-deprived or staurosporine-treated macrophages. Immunofluorescence analysis of the mitochondrial release of cytochrome c. BMDM from C57BL/6 mice were not infected or infected with L. major at a ratio of 10 parasites per BMDM, washed, and incubated for 24 h in the absence of M-CSF, staurosporine (0.5 µM) being added (Staurosporine) or not (None), during the last 2 h. Macrophages were then stained for cytochrome c release (red) and viewed under a fluorescence microscope. Punctate red staining, Mitochondrial localization; diffuse red staining, cytosolic localization. Arrows show cells with typical mitochondrial cytochrome c release.



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  		Figure 5. L. major infection prevents caspase-3 processing in staurosporine-treated macrophages. BMDM were treated as described in Figure 4 . Uninfected (lane 1) and L. major-infected (lane 2) BMDM in the absence of staurosporine treatment and L. major-infected (lane 3) and uninfected (lane 4) BMDM after staurosporine treatment were analyzed by Western blotting for caspase-3 cleavage. Tubulin was used as a control of loading.

Inhibitors of NF-{kappa}B activation do not suppress L. major-mediated prevention of PCD
Antiapoptotic members of the Bcl-2 protein family exert their preventive effect on PCD induction by repressing mitochondrial outermembrane permeabilization and cytochrome c release [57 ]. In macrophages, activation of the transcription factor NF-{kappa}B has been reported to play an important role in maintenance of mitochondria homeostasis and cell survival by up-regulating the expression of a Bcl-2-related, antiapoptotic protein A1 [58 , 59 ]. In another cell type, the intracellular parasite Theileiria parva has been reported to prevent PCD induction in the infected cell by activating the NF-{kappa}B pathway [21 ]. Therefore, we investigated whether NF-{kappa}B activation may be involved in L. major-mediated prevention of BMDM death by using two inhibitors of NF-{kappa}B, the proteasome inhibitor MG-132, which acts by preventing inhibitor of {kappa}B (I{kappa}B) denaturation [59 ], and sesquiterpene lactone PL, which acts by targeting the I{kappa}B kinase [60 ]. As shown in Figure 6 , PL induced a major increase in BMDM death induced by M-CSF deprival but had no effect on the survival of M-CSF-deprived, L. major-infected BMDM. MG-132 had no or only a minor effect on cell death induced by M-CSF deprival, whether in the absence or presence of L. major infection (Fig. 6) . These findings suggested that NF-{kappa}B activation might play a role in macrophage survival by repressing, to some extent, PCD induction in response to M-CSF deprival but that L. major infection can still exert a significant, preventive effect on the BMDM PCD induced by M-CSF deprival, even in the presence of NF-{kappa}B inhibitors. Thus, our findings imply that L. major-mediated repression of PCD does not require NF-{kappa}B-dependent transcription of antiapoptotic genes.



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  		Figure 6. NF-{kappa}B inhibitors do not suppress L. major-mediated prevention of macrophage death. BMDM from C57BL/6 mice were incubated for 1 h in the absence (–) or presence of the proteasome inhibitor MG-132 (10 µM), which inhibits NF-{kappa}B by preventing I{kappa}B denaturation, or with sesquiterpene lactone PL (10 µM), which inhibits NF-{kappa}B by targeting the I{kappa}B kinase complex and were then infected (L. major infection 10/1) or not (No infection) with L. major. BMDM were then incubated for 24 h in the presence (+M-CSF) or absence (M-CSF deprival) of M-CSF, and flow cytometry analysis using DiOC6 was performed to assess {Delta}{psi}m loss. Open and shaded bars, Uninfected BMDM; solid bars, L. major-infected BMDM. Results show one experiment representative of two independent experiments.


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DISCUSSION
 
Ours findings indicate that L. major-infected BMDM show enhanced survival in two situations that induce PCD in all nucleated mammalian cells, deprival of survival factors [2 , 42 ], and treatment with the protein kinase inhibitor staurosporine [38 ]. This delaying effect on PCD induction was observed in BMDM from the BALB/c mice, which develop a Th2 cytokine response to L. major in vivo, resulting in parasite dissemination and lethal disease [34 , 37 ], and the C57BL/6 mice, which develop a Th1 cytokine response to L. majorin vivo, resulting in immune-mediated control of parasite dissemination and recovery from disease [34 , 37 ]. Therefore, our findings suggest that L. major-mediated repression of macrophage PCD is an intrinsic property of the parasite that does not depend on the host-specific, genetic control of Th1/Th2 cytokine secretion. This confirms and extends the previous observation that another Leishmania species, L. donovani, prevents oligonucleosomal DNA fragmentation [22 ] (a dispensable feature of PCD) [36 ] in BMDM from Leishmania-susceptible BALB/c mice upon deprival of M-CSF. Together, our findings and this previous observation [22 ] support the idea that the enhancement of macrophage survival, through repression of PCD, may be a common property of all parasites belonging to the Leishmania species.

It has been proposed that L. donovani may repress BMDM PCD induced by M-CSF deprival through the secretion of TNF-{alpha} by the infected macrophages [22 ]. TNF-{alpha} is a member of the TNF ligand family that is expressed on the cell surface or released as a soluble cytokine and can induce apoptosis or cell differentiation and/or proliferation through the engagement of the cell-surface TNF-{alpha} receptors [49 ]. TNF-{alpha} is one of the cytokines secreted by macrophages in response to various pathogens [61 ], and TNF-{alpha} has been reported to act as a survival factor in human monocytes [45 ]. Our findings indicate, however, that the preventive effect of L. major on murine BMDM PCD does not involve TNF-{alpha}-mediated signaling. Fas ligand (CD95L) is another member of the TNF ligand family that can induce, upon engagement of its CD95 receptor, PCD [48 ] or cell differentiation and/or proliferation [46 ]. However, our findings also indicate that CD95-mediated signal transduction is neither involved in BMDM death induced by M-CSF deprival nor in the preventive effect by L. major infection on BMDM death.

Death signaling, resulting from the engagement by their ligands of the cell-surface death receptors of the TNFR family, represents one of the rare instances in which activation of the effector caspases, such as caspase-3, involved in the execution of apoptosis, can occur independently of the participation of mitochondria through the direct activation of the caspase cascade downstream of the death receptor engagement [50 , 52 ]. Almost all other proapoptotic stimuli, including staurosporine [53 ], require a mitochondria-dependent step, controlled by members of the Bcl-2/Bax protein family, leading to mitochondrial outer membrane permeabilization and to release in the cytosol of mitochondrial intermembrane space proteins such as cytochrome c, which will trigger caspase activation [51 , 54 ]. We observed that L. major infection of BMDM results in a prevention of mitochondrial release of cytochrome c in the cytosol and activation of the effector caspase-3. Consistent with our observation of a lack of involvement of cell-surface death receptors of the TNFR family, our findings strongly suggest that L. major infection exerts its preventive effect on effector caspase activation and on cell death induction by repressing mitochondrial outermembrane permeabilization.

In macrophages, the prevention of mitochondrial outer membrane permeabilization and of mitochondria-dependent cell death has been reported to involve the expression of one member of the antiapoptotic Bcl-2 protein family, A1, through the activation of the NF-{kappa}B transcription factor [58 , 59 ]. It is interesting that in another cell type, the T lymphocyte, the intracellular parasite T. parva has been reported to prevent cell death through a pathway that involves activation of NF-{kappa}B-dependent transcription [21 ]. Therefore, it was tempting to speculate that L. major infection may promote BMDM survival through a similar NF-{kappa}B-dependent pathway. Using two different inhibitors of NF-{kappa}B activation, we observed that one inhibitor increased BMDM death induced by M-CSF deprival but that none affected the capacity of L. major to prevent death in a large proportion of the BMDM. Thus, although our findings are consistent with findings indicating that the NF-{kappa}B pathways play a general role in macrophage survival, they also suggest that NF-{kappa}B activation does not account for the selective, preventive effect exerted by L. major infection on BMDM death.

One reported effect of L. major infection on signal transduction in macrophages is the inhibition of protein kinase C (PKC) activity [62 ]. As staurosporine, a broad protein kinase inhibitor, induced BMDM death and as L. major infection prevented staurosporine-induced BMDM death, it is, however, unlikely that L. major infection promotes BMDM survival through inhibition of PKC. There are at least three potential mechanisms that may account for the repression of BMDM mitochondrial permeabilization induced by L. major infection that we observed. The first one is the induction of a NF-{kappa}B-independent transcription process, leading to the expression of antiapoptotic members of the Bcl-2 protein family. The second one is the post-transcriptional modification, such as alternative splicing, or post-translational modification, such as phosphorylation, of proapoptotic members of the Bax family [57 ]. Finally, although no gene sequence homology with the Bcl-2 family has yet been identified in any unicellular eukaryote [7 , 63 ], it cannot be excluded at this stage that antiapoptotic proteins encoded and expressed or secreted by L. major and sharing functional similarity with Bcl-2 may account for the preventive effect of L. major infection on PCD. Indeed, it should be noted that a protein with no sequence homology with any member of the Bcl-2 family that prevents PCD by controlling mitochondria permeabilization has been identified recently in a virus, the cytomegalovirus [64 ].

Although the precise mechanism of the antiapoptotic effect of L. major infection remains to be assessed, our findings have potential implications concerning the pathogenesis of this parasite. Indeed, they suggest that the capacity of L. major to delay PCD induction in its target cell, the macrophage, may play a role in host invasion and in the persistence of the parasite in the infected cells. Recently, L. major infection was reported to also delay PCD induction in vitro and in vivo in another cell target, the polymorphonuclear neutrophil granulocyte [65 ]. Although the precise mechanism involved and in particular, the potential involvement of mitochondria and cytochrome c release were not investigated in this study, inhibition of PCD was also found to be associated with a reduction in caspase-3 activity [65 ]. Thus, these data together with our findings suggest that inhibition of caspase-3 processing and activation is a broad effect of L. major infection in at least two different cell types. More generally, together with previous findings indicating that Toxoplasma gondii [23 24 25 ] and T. parva [21 ] also repress PCD induction in the infected cells, our findings support the hypothesis that several intracellular parasites may have evolved strategies favoring the survival of their host cells.

In summary, our findings indicate that L. major has the capacity to enhance the survival of the macrophages it infects by preventing effector caspase activation in response to proapoptotic stimuli, through a mechanism that appears to act at the level of the macrophage mitochondria, by preventing mitochondrial outer membrane permeabilization and cytochrome c release. Further investigations will be required to elucidate the molecular mechanism(s) allowing Leishmania to prevent PCD in its obligate host cell. Finally, Leishmania-mediated modulation of macrophage survival should provide a useful model to explore to what extent the capacity to repress PCD may play an important and general role in host/parasite interactions, by favoring invasion, persistence, and virulence of intracellular parasites.


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ACKNOWLEDGEMENTS
 
We thank Dr. Y. J-F. Garin (Saint Louis Hospital, France) for providing L. major, Dr. P. Vandenabeele (Ghent, Belgium) for the gift of antibody raised against mouse caspase-3, and F. Petit for technical advice. The research programs of EMI-U9922 are supported by institutional funding from INSERM, Université Paris 7, AP-HP, and by grants from Agence Nationale de Recherche sur le SIDA (ANRS), Ensemble Contre le Sida (ECS), Université Paris 7 Valorisation, Fondation de la Recherche Medicale (FRM), Etablissement Français des Greffes (to J. C. A.), a postdoctoral fellowship from ECS (to K. A.), and doctoral fellowships from Direction Générale pour l’Armement and FRM (to D. A.).


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FOOTNOTES
 
1 Current address: Université Cadi Ayyad, Faculté Polydisciplinaire, BD de Quachla, Sidi Bouzid, Safi, Morocco. Back

Received October 10, 2001; revised January 27, 2004; accepted February 4, 2004.


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