ABSTRACT
Leishmania parasites alternate between flagellated promastigotes in sand flies and nonflagellated amastigotes in mammals, causing a spectrum of serious diseases. To survive, they must resist the harsh conditions in phagocytes (including acidic pH, elevated temperature, and increased oxidative/nitrosative stress) and evade the immune response. Recent studies have highlighted the importance of sphingolipid (SL) metabolism in Leishmania virulence. In particular, we have generated a Leishmania major iscl − mutant which is deficient in SL degradation but grows normally as promastigotes in culture. Importantly, iscl − mutants cannot induce pathology in either immunocompetent or immunodeficient mice yet are able to persist at low levels. In this study, we investigated how the degradation of SLs might contribute to Leishmania infection. First, unlike wild-type (WT) L. major, iscl − mutants do not trigger polarized T cell responses in mice. Second, like WT parasites, iscl − mutants possess the ability to downregulate macrophage activation by suppressing the production of interleukin-12 (IL-12) and nitric oxide. Third, during the stationary phase, iscl − promastigotes were extremely vulnerable to acidic pH but not to other adverse conditions, such as elevated temperature and oxidative/nitrosative stress. In addition, inhibition of phagosomal acidification significantly improved iscl − survival in murine macrophages. Together, these findings indicate that SL degradation by Leishmania is essential for its adaption to the acidic environment in phagolysosomes but is not required for the suppression of host cell activation. Finally, our studies with iscl − mutant-infected mice suggest that having viable, persistent parasites is not sufficient to provide immunity against virulent Leishmania challenge.
INTRODUCTION
Leishmania parasites infect 10 million to 12 million people in 88 countries (5). During their life cycle, these protozoans alternate between flagellated promastigotes in the sand fly midgut and nonflagellated amastigotes in mammalian phagocytes. Current drugs are limited in efficacy, and no safe vaccine is available. To develop new therapeutics, it is crucial to understand the molecular mechanism by which Leishmania parasites survive within the mammalian host and cause diseases.
In many eukaryotes, sphingolipids (SLs) and their metabolites are important membrane components and signaling molecules (1). While mammalian cells mostly synthesize sphingomyelin and complex glycosylated SLs, the majority of SLs in Leishmania belong to inositol phosphorylceramide (IPC) (6, 34). Functions of SL metabolism in Leishmania have been probed using gene knockout mutants. In Leishmania major, an important role of sphingoid base synthesis and degradation is to generate ethanolamine phosphate, a metabolite essential for the proliferation and differentiation of promastigotes from noninfective, replicative procyclics to highly infective, nonreplicative metacyclics (a process called metacyclogenesis) (20, 33). The reason why ethanolamine phosphate is required for growth and metacyclogenesis is under investigation, and preliminary data suggest that it is used to synthesize ethanolamine plasmalogen (very abundant in Leishmania) and potentially other important phospholipids (33).
In addition to producing ethanolamine phosphate, SL metabolism has other important functions in Leishmania. Despite their lack of sphingomyelin synthesis, Leishmania parasites possess a potent neutral sphingomyelinase (SMase) called inositol phosphosphingolipid phospholipase C-like (ISCL), which is responsible for the degradation of both host-derived sphingomyelin and parasite-derived IPC (35). ISCL null mutants (iscl− mutants, generated from wild-type [WT] L. major) had only minor defects during the promastigote stage in culture but completely failed to multiply or cause disease in susceptible BALB/c mice (35). Importantly, this deficiency can be corrected only by other SMases and not IPC hydrolases (IPCases), suggesting that the degradation of host-derived sphingomyelin (not parasite-synthesized IPC) is essential for Leishmania growth in mammals (35).
Because ISCL is localized in mitochondria during the promastigote stage (35), it may play a role in respiration or energy production under low-sugar conditions (such as late stationary phase in culture or inside the phagolysosome of macrophages). Alternatively, the degradation of host sphingomyelin may be important for the uptake of nutrients or the adaptation to hostile conditions in the mammalian host. Furthermore, it is also possible that ISCL is required for the downregulation of host cell defense and/or the evasion of immunity. All of these possibilities (not mutually exclusive) could explain the lack of acute virulence in iscl− mutants and lead to novel insight into the role of SL degradation in Leishmania infection.
In this study, we probed the molecular mechanism by which ISCL may contribute to Leishmania virulence. First, we examined the cytokine/antibody production in mice after iscl− mutant infection to determine whether SL degradation was involved in the manipulation of the T cell response. We also characterized the interaction between iscl− mutants and murine macrophages (the definitive host cells for Leishmania) to determine whether ISCL was required for the suppression of nitric oxide (NO) and interleukin-12 (IL-12) production (7–9). Furthermore, we investigated whether ISCL was needed for resistance to elevated temperatures, acidic pHs, reactive oxygen intermediates (ROIs), and reactive nitrogen intermediates (RNIs). The ability to tolerate these harsh conditions is essential for the establishment of Leishmania infection. In total, our study aims to uncover the novel roles of SL metabolism in Leishmania virulence.
MATERIALS AND METHODS
Mice and reagents.C57BL/6 and BALB/c mice (female, 7 to 8 weeks old) were purchased from Charles River Laboratories International (Wilmington, MA). BALB/c SCID (CBySmn.CB17-Prkdcscid/J), iNOS− (B6.129P2-Nos2tm1Lau/J), and phox− (B6.129S6-Cybbtm1Din/J) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). All procedures involving mice were approved by the Animal Care and Use Committee at Texas Tech University (PHS Approved Animal Welfare Assurance no. A3629-01).
Griess reagent for NO measurement was purchased from Cayman Chemical Company (Ann Arbor, MI). Enzyme-linked immunosorbent assay (ELISA) kits to measure gamma interferon (IFN-γ), IL-4, IL-10, and IL-12p40 were purchased from eBioscience, Inc. (San Diego, CA). Fluorescein-tetramethylrhodamine (TMR) double-labeled dextran (molecular weight [MW] = 70,000) and carboxyfluorescein succinimidyl ester (CFSE) were purchased from Invitrogen Corporation. [3H]thymidine was purchased from PerkinElmer, Inc. (San Jose, CA). All other chemicals were purchased from Sigma (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA), unless otherwise specified.
Leishmania culture.Unless otherwise specified, L. major LV39 clone 5 (Rho/SU/59/P) promastigotes were cultured at 27°C in M199 (pH 7.4) medium with 10% heat-inactivated fetal bovine serum (FBS) and other supplements (11). The ISCL-null (iscl−) mutant and its reconstituted control (iscl−/+ISCL strain) were maintained in the presence of proper antibiotics (10 μg/ml of puromycin and 10 μg/ml of blasticidin for iscl− mutants and 20 μg/ml of G418 for iscl−/+ISCL strains) as previously described (35). Cell growth over time was determined using a hemacytometer. Cell viability was determined by flow cytometry after cells were stained with propidium iodide (34). To generate soluble Leishmania antigen (SLA), L. major wild-type (WT) parasites (1 × 108 cells/ml in phosphate-buffered saline) were subjected to three freeze-thaw cycles, followed by sonication for 5 min on ice, and then stored in aliquots at −80°C.
Mouse footpad infection.To evaluate Leishmania virulence, mice were infected in their left footpads by subcutaneous injection (5 to 6 mice per group) with day 3 stationary-phase promastigotes (3 days after reaching maximal density in culture) of the WT, iscl− mutant, or iscl−/+ISCL strain as previously described (26, 35). To determine whether iscl− mutants can provide protection against virulent challenge, BALB/c mice were first infected with iscl− mutants in their right footpads with or without adjuvant (CpG [5′-TCC ATG ACG TTC CTG ACG TT-3′] or non-CpG [5′-TCC AGG ACT TCT CTC AGG TT-3′] oligonucleotide; 50 μg/mouse) for 8 weeks. These mice and age-matched naïve mice were then infected with WT parasites in their counterlateral (left) footpads. As a control, BALB/c mice were also vaccinated with lpg2− parasites (1 × 107 each) for 4 weeks in their right footpads and then challenged with WT cells (1 × 106 each) in their counterlateral footpads. Lesion sizes were measured with a Vernier caliper, and parasite numbers in their infected footpads were determined by limiting dilution assay as described previously (25, 26).
Murine macrophage infection and measurement of NO/IL-12p40 production.Bone marrow-derived macrophages were generated from mouse femur cells in complete Dulbecco modified Eagle medium (DMEM)-10% FBS plus 20 ng/ml of recombinant macrophage colony-stimulating factor at 37°C with 5% CO2. Macrophage infection was performed using promastigotes opsonized with C57BL/6 mouse serum at a multiplicity of infection (MOI) of 15:1 as previously described (17, 23). To examine the effect of lysosomal acidification inhibitor on Leishmania infection, bone marrow macrophages (from BALB/c mice) were treated with 100 nM bafilomycin A1 (BAF) or 5 μM chloroquine phosphate for 30 min. The drug was then washed off, and Leishmania infection was performed as described above. To determine NO and IL-12p40 production, promastigotes were allowed to infect macrophages for 4 h, and then macrophages were washed and transferred to media containing various concentrations of lipopolysaccharide (LPS) and IFN-γ (0 to 50 ng/ml for each). After overnight incubation, concentrations of NO and IL-12p40 in the supernatant were determined using the Griess reagent and IL-12p40 ELISA kit, respectively.
Analysis of phagosomal acidification by fluorescein-TMR labeling.To confirm the effect of BAF on lysosomal acidification, BALB/c bone marrow macrophages were incubated in 0.2 mg/ml of TMR-fluorescein dextran for 1 h. Cells were then washed twice with DMEM, and half were treated with 100 nM BAF for 30 min, followed by incubation at 37°C in 5% CO2 and DMEM-10% FBS. After 2, 24, or 48 h, macrophages were stained with Hoechst 33242 dye and observed using an Olympus BX51 fluorescence microscope. To assess the lysosomal acidification of parasitized host cells, macrophages were infected with WT promastigotes (MOI = 15:1) for 2 h and then labeled with TMR-fluorescein dextran as described above.
Lymphocyte proliferation assay and quantitation of cytokine production.Mice were sacrificed, and single-cell suspensions from draining popliteal lymph nodes (dLNs) were prepared. Lymphocyte numbers were counted after lymphocytes were stained with 0.4% trypan blue and then cultured in DMEM supplemented with 10% FBS, 2 mM glutamine, 1× penicillin-streptomycin, 1 mM sodium pyruvate, and 50 μg/ml gentamicin in a 37°C/5% CO2 incubator. To assess lymphocyte proliferation by [3H]thymidine incorporation assay, dLN cells were cultured in 96-well plates (4 × 106 cells/ml) and stimulated with SLA (equivalent to 8 × 106 parasites/ml) or anti-CD3 monoclonal antibody (1 μg/ml) (28). After 72 h, 0.5 μCi of [3H]thymidine was added to each well, and after 16 to 18 h, cells were harvested to count radioactivity in a liquid scintillation counter. Lymphocyte proliferation potential was also evaluated by CFSE assay. Briefly, isolated lymphocytes (1 × 106 cells/ml) were incubated in 5 μM CFSE at 37°C and 5% CO2 for 10 min, followed by the addition of a 5× volume of ice-cold medium to quench the staining. Cells were then washed 3 times with phosphate-buffered saline and seated in 96-well round-bottom plates for 5 days before flow cytometry (488-nm excitation).
To measure cytokine production, lymphocytes from dLNs were cultured in 24-well plates (4 × 106 cells/ml) and stimulated with SLA (equivalent to 8 × 106 parasites/ml) for 72 h. Supernatants were assayed for IFN-γ, IL-4, or IL-10 using appropriate ELISA kits (29).
Serum antibody titer.Serum samples were collected from naïve and infected mice at the indicated times. To assess Leishmania-specific antibody, Immulon plates (Thermo Electron, Milford, MA) were coated with promastigote lysates (50 μg/ml) overnight at 4°C. After blocking, plates were incubated with individual serum samples (1:50 to 1:200 dilution) for 1 h at room temperature. Then, plates were incubated with horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG1 or IgG2a (1:1,000; BD Biosciences, San Jose, CA) secondary antibodies. Plates were developed with the tetramethylbenzidine (TMB) substrate (BD Biosciences), and optical density (OD) values at 450 nm were measured with a Multiskan Ascent ELISA reader (Labsystems, Helsinki, Finland).
Tolerance of Leishmania parasites to elevated temperatures, acidic pHs, oxidants, and nitrosative stress.Promastigotes were cultured in regular medium (M199 with supplements, pH 7.4) at 27°C, and either log-phase (1 × 106 to 5 × 106 cells/ml) or day 1 stationary-phase (2 × 107 to 2.5 × 107 cells/ml) promastigotes were used in the following assays. To test temperature sensitivity, cells were kept in regular medium at either 27°C or 37°C with 5% CO2. For pH assay, parasites were cultured at 27°C in either a regular medium (pH 7.4) or an acidic medium (same as the regular medium except that the pH was adjusted to 5.0 with HCl) at the same density. To measure sensitivity to oxidative and nitrosative stress, parasites were cultured in 24-well plates (regular medium, 27°C) in the presence of various concentrations of H2O2 or S-nitroso-N-acetylpenicillamine (SNAP) (15). Cell density and viability were determined as indicated in Fig. 6.
Statistical analysis.The difference between the two groups was determined by Student's t test using SigmaPlot 11.0 (Systat Software Inc., San Jose, CA). P values indicating statistical significance were grouped into values of <0.05 and <0.01.
RESULTS
iscl− mutants failed to induce pathology in immunocompetent or immunocompromised mice.BALB/c mice are susceptible to L. major infection due to a Th2-biased response leading to uncontrolled parasite growth and severe pathology, whereas C57BL/6 mice are resistant due to a protective Th1-biased response (19). We have shown that iscl− mutants cannot induce any significant lesions (<0.1 mm) in BALB/c mice but can persist at low levels for several months (35) (Fig. 1A and B). Here we tested if iscl− mutants could cause pathology or persist in resistant C57BL/6 mice. While L. major WT and iscl−/+ISCL parasites induced moderate lesions (less severe than those of infected BALB/c mice) which gradually resolved over time, iscl− mutants failed to cause any significant pathology (lesions of <0.2 mm) in C57BL/6 mice (Fig. 1C). As seen with the BALB/c mouse infection, lesion sizes correlated with parasite numbers in their footpads and iscl− mutants persisted at low levels (102 to ∼103cells/infected footpad) in C57BL/6 mice for more than 4 months (Fig. 1D).
iscl− mutants failed to induce pathology in immunocompetent or immunocompromised mice but persisted at low levels. BALB/c (A and B), C57BL/6 (C and D), or BALB/c SCID (E and F) mice were infected in their footpads with stationary-phase promastigotes of WT, iscl−, or iscl−/+ISCL parasites (1 × 106 parasites/mouse, 5 to 6 mice per group). Lesion sizes were measured weekly with a caliper and are recorded in panels A, C, and E (•, WT; ○, iscl− parasites; ▾, iscl−/+ISCL parasites). Parasite numbers in their infected footpads were determined by limiting dilution assay and are summarized in panels B, D, and F (black bars, WT; white bars, iscl− mutant; gray bars, iscl−/+ISCL strain). Error bars represent standard deviations (*, P < 0.05; **, P < 0.01).
Next, we used the immunocompromised SCID mice (BALB/c background) to determine whether adaptive immunity plays a major role in the control of iscl− mutants. Clearly, iscl− mutants failed to cause pathology or propagate to the same levels as WT and iscl−/+ISCL parasites in SCID mice (Fig. 1E and F). Therefore, iscl− mutants cannot proliferate efficiently or cause disease in mammals even in the absence of T cell and B cell functions.
iscl− mutants did not trigger any polarized T cell responses in mice.To understand the immune response induced by iscl− mutants, we first examined the proliferative capacity of lymphocytes from infected BALB/c mice. On day 3 postinfection, the draining lymph nodes (dLNs) from iscl− mutant-infected mice contained numbers of cells (6.5 × 107 cells/dLN) similar to those from WT- and iscl−/+ISCL strain-infected animals (7.5 × 107 cells/dLN) (Table 1). Meanwhile, the proliferative potentials of lymphocytes were comparable among WT-, iscl− mutant-, and iscl−/+ISCL strain-infected mice after stimulation with SLA (27) (Fig. 2A), suggesting that iscl− mutants interacted with the host immune system normally during the early stage of infection. This was consistent with the WT-like survival rate of iscl− mutants in mice at 3 days postinfection (Fig. 1B). However, at 8 weeks postinfection, the dLNs from iscl− mutant-infected mice became much smaller and contained fewer cells (2.78 × 106 cells/dLN) than the ones from WT- or iscl−/+ISCL strain-infected mice (4 × 107 to 6.25 × 107 cells/dLN) (Table 1). In addition, proliferation assays showed that lymphocytes from iscl− mutant-infected mice did not respond strongly to SLA but were still stimulated by an anti-CD3 antibody (indicative of functional dLN cells that were not responsive to Leishmania) (Fig. 2B). This diminished lymphocyte response seemed to correlate with the low parasite numbers in iscl− mutant-infected mice (Fig. 1B).
iscl− mutants stimulated transient inflammatory and proliferative responses. BALB/c mice infected with the WT, iscl− mutant, or iscl−/+ISCL strain were sacrificed at 3 days (A, C to G) or 8 weeks (B) postinfection (P.I.). (A and B) Draining lymph nodes (dLNs) were isolated, and the proliferative potentials of lymphocytes were determined by [3H]thymidine incorporation assay. Black bars, unstimulated; white bars, anti-CD3 monoclonal antibody; gray bars, Leishmania soluble antigen (SLA). (C to E) Infected BALB/c mice were sacrificed at 3 days postinfection, and dLN cells were stimulated with SLA. The amounts of IFN-γ (C), IL-4 (D), and IL-10 (E) in culture supernatants were determined, and the ratios of values for SLA-treated to values for unstimulated cells were calculated for each cytokine. (F and G) IL-4/IFN-γ and IL-10/IFN-γ ratios are determined from the SLA-treated samples. Error bars represent standard deviations.
Number of cells in the dLNs of infected mice
Next, we questioned if the lack of iscl− mutant proliferation in mice was associated with an elevated Th1 response. The production of IFN-γ, IL-4, and IL-10 from dLN cells was measured after SLA stimulation (Fig. 2C to G and 3). To offset potential variations in dLN cell numbers, we calculated the ratios of values after SLA stimulation to values without stimulation for each cytokine analysis. At day 3 postinfection, BALB/c lymphocytes did not produce much more IFN-γ but did generate 3 to 10 times more IL-4 and IL-10 upon SLA stimulation (Fig. 2C to E). At this early stage, there was no major bias toward either Th1 or Th2 and no significant difference between WT- and iscl− mutant-infected mice (Fig. 2F and G). However, at 8 weeks postinfection, WT and iscl−/+ISCL parasites triggered high levels of IL-4 and IL-10 in BALB/c mice while iscl− mutants failed to do so (Fig. 3B and C). Meanwhile, both the mutant and control parasites induced low levels of IFN-γ (Fig. 3A). In C57BL/6 mice, iscl− mutants did not stimulate the production of IFN-γ, which was prominent in WT and iscl−/+ISCL strain infections (Fig. 3A). Expression levels of IL-4 and IL-10 were much lower than in BALB/c mouse infections, and there was no significant difference between iscl− mutants and control parasites (Fig. 3B and C). Based on the values of IL-4/IFN-γ and IL-10/IFN-γ, WT and iscl−/+ISCL parasites induced a Th2-biased response in BALB/c mice and a Th1-dominated response in C57BL/6 mice (as expected), whereas iscl− mutants failed to trigger any significant T cell responses (Fig. 3D and E). We also performed the same cytokine analyses at 4 and 12 weeks postinfection and obtained results very similar to those from the 8-week experiment (data not shown). Together, these data suggest that the limited number of iscl− mutants fails to adequately engage host immunity to induce the type of T cell response seen in WT-infected mice.
iscl− mutants failed to induce polarized T cell responses in mice. BALB/c or C57BL/6 mice were infected in their footpads and sacrificed after 8 weeks. Lymphocytes were isolated from dLNs and plated on 96-well dishes. After stimulation with SLA for 3 days, culture supernatants were collected to measure the level of IFN-γ (A), IL-4 (B), and IL-10 (C). (A to C) SLA-treated/unstimulated cell ratios were calculated for each cytokine. (D and E) IL-4/IFN-γ and IL-10/IFN-γ ratios (both from SLA-treated samples). Error bars represent standard deviations (*, P < 0.05; **, P < 0.01).
Furthermore, we measured the level of anti-Leishmania antibody (IgG) in the sera of infected mice. At 8 weeks postinfection, both WT and iscl− parasites triggered robust production of IgG1/IgG2a in BALB/c mice but not in C57BL/6 or SCID mice (see Fig. S1 in the supplemental material). However, the level of IgG1/IgG2a in iscl− mutant-infected BALB/c mice subsided significantly during long-term persistence (56 weeks postinfection) (Fig. S1), suggesting that the limited number of persistent iscl− parasites was unable to sustain antibody production.
iscl− mutant infection did not confer protection against virulent challenge.The cytokine production profile of iscl− mutant-infected BALB/c mice was similar to what was observed previously using another L. major mutant, an lpg2− mutant, which is defective in the synthesis of mannose-containing phosphoglycans (28). Like iscl− parasites, lpg2− parasites failed to cause disease and were able to persist in BALB/c mice (24). Previous reports (12, 28) and our own studies (see Fig. S2 in the supplemental material) have demonstrated that those asymptomatic, lpg2− parasite-infected mice were protected from secondary challenge with WT L. major parasites. To determine if the persistent iscl− mutant can also provide protection, BALB/c mice were infected with iscl− mutants in the presence or absence of CpG oligonucleotide as an adjuvant (18). Eight weeks later, the infected and age-matched naïve mice were challenged with WT parasites in their counterlateral footpads. As shown in Fig. 4A and B, all mice exhibited normal lesion progression and contained similar numbers of parasites at 3 or 8 weeks after the virulent challenge. We also measured the production of IFN-γ, IL-4, and IL-10 from dLN cells and calculated the ratios of IL-4 to IFN-γ (Fig. 4C) and IL-10 to IFN-γ (Fig. 4D). Clearly, all groups of mice expressed higher levels of Th2 cytokines than Th1 cytokine, regardless of whether they were “vaccinated” with iscl− mutants. Therefore, iscl− mutants (with or without CpG adjuvant) could not provide protection against virulent challenge despite their ability to persist and induce antibody production in mice (Fig. S1).
iscl− mutant-infected BALB/c mice were not protected against virulent challenge. (A) Age-matched naïve mice (•) or those “vaccinated” (Vac.) with iscl− parasites (1 × 107 each) (○, iscl− parasites only; ▾, iscl− parasites + non-CpG; Δ, iscl− parasites + CpG) for 8 weeks were challenged with WT parasites (1 × 106 each) in their counterlateral footpads. Lesions were measured over time. (B) Parasite numbers in the WT-parasite-infected footpads were determined at 3 weeks (black bars) or 8 weeks (white bars) postchallenge. Also at 8 weeks postchallenge, cytokine production from dLN cells was measured, and the ratios of IL-4 to IFN-γ (C) and IL-10 to IFN-γ were determined (D). Error bars represent standard deviations.
iscl− mutants did not activate murine macrophages.Since iscl− parasites do not stimulate a strong antileishmanial immune response in mice, their lack of virulence is likely due to deficiency in growth/replication in the mammalian host. We previously reported that iscl− mutants were unable to multiply in BALB/c macrophages (35). It was not clear whether iscl− mutants were hypersensitive to mammalian culture conditions or unable to suppress macrophage activation. To address this question, we first tested whether iscl− mutants could replicate in macrophages in the absence of reactive oxygen intermediates (ROIs) or reactive nitrogen intermediates (RNIs). As expected, in C57BL/6 macrophages (derived from bone marrow cells), iscl− mutants survived poorly compared to WT and iscl−/+ISCL parasites after 24 h of infection (Fig. 5A and B). Importantly, very similar results were observed using macrophages from iNOS− mice (deficient in inducible nitric oxide synthase [31]) (Fig. 5C and D) and phox− mice (deficient in NADPH oxidase) (10) (Fig. 5E and F). Therefore, even in the absence of ROI/RNI activity, iscl− mutants remain defective in intracellular growth.
iscl− parasites survived poorly in murine macrophages (MΦs) but still suppressed host cell activation. (A to F) Stationary-phase promastigotes (•, WT; ○, iscl− mutant; ▾, iscl−/+ISCL strain) were used to infect bone marrow-derived murine macrophages from C57BL/6 (A and B), iNOS− (C and D), or phox− (E and F) mice. Percentages of infected murine macrophages (A, C, and E) and the numbers of parasites per 100 murine macrophages (B, D, and F) were recorded. As a control in each experiment, WT parasites were used to infect murine macrophages that were activated with 100 ng/ml of LPS and 100 ng/ml of IFN-γ (▵). (G and H) BALB/c murine macrophages were infected by stationary-phase promastigotes (black bars, uninfected parasites; white bars, WT parasites; gray bars, iscl− parasites; hashed bars, iscl−/+ISCL parasites), followed by overnight activation using various concentrations of LPS and IFN-γ. Concentrations of NO (G) and IL-12p40 (H) in culture supernatants were measured. Error bars represent standard deviations (*, P < 0.05; **, P < 0.01).
As a control in these experiments, WT parasites failed to propagate in macrophages activated with LPS and IFN-γ (Fig. 5A to F). To examine if iscl− mutants can invade host cells silently, we measured the levels of NO and IL-12p40 in the supernatant of infected macrophages (Fig. 5G and H, no LPS/IFN-γ), and results showed that iscl− mutants maintained the ability to enter macrophages without triggering the overproduction of NO or IL-12 (a T cell-stimulating factor). To determine if intracellular iscl− parasites can suppress the activation of host cells, Leishmania-infected macrophages were transferred to medium containing various concentrations of LPS and IFN-γ (6 to 50 ng/ml each). As shown in Fig. 5G and H, uninfected macrophages produced relatively high levels of NO and IL-12p40, whereas both WT and iscl− parasites inhibited the stimulatory effect (by 2- to 3-fold) from LPS/IFN-γ. Therefore, ISCL is not required for the silent entry or suppression of macrophages.
iscl− mutants were vulnerable to acidic pH but not to heat or oxidative or nitrosative stress in stationary phase.Residing in the phagolysosome of macrophages, Leishmania parasites must adapt to a hostile environment characterized by acidic pH, elevated temperature (compared to that of the sand fly stage), and increased oxidative/nitrosative stress (from ROIs/RNIs). Here we investigated whether ISCL was required for Leishmania to survive these conditions. Because iscl− amastigotes were difficult to obtain (due to their inability to replicate in mice), we used stationary-phase promastigotes which contained the virulent metacyclic forms (20). To examine if ISCL is required for pH tolerance, parasites were cultured in either the regular, neutral medium (pH 7.4) or an acidic medium (same as the neutral medium except that the pH was adjusted to 5.0). Cell growth and viability were monitored daily by direct counting and flow cytometry, respectively (Fig. 6). As we have shown before (35), under neutral pH, 30 to 50% of iscl− mutants and 10 to 20% of control cells were dead after 3 days in stationary phase (Fig. 6A and B). This defect appeared to be greatly exacerbated at pH 5.0, under which condition the iscl− parasites showed 80 to 100% death (versus 25 to 50% for WT and iscl−/+ISCL parasites) after 3 days in stationary phase (Fig. 6C and D). When log-phase promastigotes were inoculated into pH 5.0 medium, iscl− mutants showed a growth rate comparable to that of WT parasites (see Fig. S3 in the supplemental material) but died quickly after entering stationary phase, similar to what we observed in the experiment reported in Fig. 6. Therefore, ISCL is clearly involved in the resistance to acidic pH in stationary phase, which could be responsible for the poor survival of iscl− parasites in macrophages.
Ability of iscl− mutants to survive under acidic pH. Stationary-phase promastigotes were cultured at 2.0 × 107 cells/ml under pH 7.4 (A and B) or pH 5.0 (C and D). Black bars, WT parasites; white bars, iscl− parasites; gray bars, iscl−/+ISCL parasites. Cell density (A and C) and viability (B and D) (PI, propidium iodide) were measured at 0 to 4 days in stationary phase. Experiments were repeated three times, and error bars represent standard deviations (*, P < 0.05; **, P < 0.01).
To determine if ISCL is required for thermotolerance, stationary-phase cells were incubated at either 27°C (temperature for regular promastigote culture) or 37°C with 5% CO2 (the condition for mammalian cell culture). Results showed that parasites survived poorly at 37°C compared to 27°C (see Fig. S4 in the supplemental material). However, the difference between iscl− mutants and control cells at 37°C was not as significant as what was observed under acidic pH (Fig. 6 and see Fig. S4). Therefore, it seems unlikely that ISCL plays any major roles in thermotolerance.
We also tested whether iscl− mutants were hypersensitive to oxidative or nitrosative stress by inoculating stationary-phase promastigotes in the presence of H2O2 or SNAP, a NO-releasing agent. As summarized in Fig. S5 in the supplemental material, parasites showed a dosage-dependent response to both H2O2 and SNAP; notably, iscl− mutants were similar in culture density, having a better rate of survival than WT and iscl−/+ISCL parasites. Thus, ISCL is not required for the resistance to ROIs or RNIs. As expected, stationary-phase cells were more tolerant to oxidative and nitrosative stress than log-phase cells (data not shown).
Inhibition of phagosomal acidification improved iscl− parasites' survival in macrophages.To examine whether the hypersensitivity to acidic pH is responsible for the poor survival of iscl− mutants in mammalian cells, we treated macrophages with a lysosomal V-type H+-ATPase inhibitor, bafilomycin A1 (BAF) (2). The effect of BAF on lysosomal acidification was demonstrated using the pH-sensitive fluorescein-TMR double-labeled dextran. As illustrated in Fig. S6A and S7B in the supplemental material, the fluorescein-TMR dextran taken up by control macrophages lost most of its green fluorescence in 24 h, indicative of efficient lysosomal acidification. In contrast, BAF treatment of macrophages clearly blocked the acidification process (Fig. S6B and S7B). Significantly, pretreatment of macrophages with BAF also improved the survival of iscl− mutants to a level close to that of WT parasites (Fig. 7). Very similar results were observed when macrophages were pretreated with chloroquine, another drug known to prevent lysosomal acidification (data not shown). It is worth noting that in addition to having an effect on lysosomal pH, BAF and chloroquine impair phagosome maturation (3). Indeed, WT parasites also survived a little better in BAF- or chloroquine-treated macrophages (Fig. 7 and data not shown), suggesting that phagosomal acidification and maturation are important for the control of intracellular pathogens.
iscl− parasites survived better in murine macrophages (MΦs) pretreated with bafilomycin (BAF) than in untreated macrophages. Bone marrow murine macrophages from BALB/c mice were treated with 100 nM BAF for 30 min before infection. Stationary-phase promastigotes were used to infect naïve (•, WT parasites; ▾, iscl− parasites) or treated (○, WT parasites; ▵, iscl− parasites) murine macrophages. Percentages of infected murine macrophages (A) and the numbers of parasites per 100 murine macrophages (B) were recorded. Experiments were repeated twice, and error bars represent standard deviations.
Lastly, we examined the timing of phagosome acidification after L. major infection. A previous report suggests that Leishmania donovani promastigotes can delay the acidification of phagosomes for at least 24 h (30). Here, macrophages were infected with WT parasites for 2 h and then labeled with fluorescein-TMR dextran. As shown in Fig. S7A in the supplemental material, the green fluorescein signal was significantly reduced after 24 h, similar to what was observed with uninfected macrophages, although a little less dramatic (normalized in Fig. S7B). Therefore, phagosomal acidification can occur in L. major-infected host cells and negatively affect the survival of iscl− mutants. Together, our results indicate that the lack of acid tolerance may account for the poor viability of iscl− mutants in macrophages.
DISCUSSION
Persistent iscl− mutants do not trigger a strong immune response or provide protective immunity in mice.The avirulence phenotype exhibited by iscl− mutants prompted us to investigate how SL degradation may affect parasite growth in mammals. Clearly, the lack of pathology is not due to hypersensitivity to host adaptive immunity, as iscl− mutants failed to grow or cause disease even in immunocompromised SCID mice (Fig. 1E and F). In immunocompetent BALB/c mice, the numbers of dLN cells and their proliferative potentials at 3 days postinfection were similar in WT- and iscl− mutant-infected animals (Table 1 and Fig. 2A), suggesting that the loss of ISCL does not alter the early inflammatory response to L. major. However, at 8 weeks postinfection, iscl− mutant-infected mice had much fewer cells in their dLNs than WT-infected mice, and their lymphocytes did not proliferate well after SLA treatments (Fig. 2B). This result suggests that the limited number of iscl− parasites (<100 cells/mouse at 8 weeks postinfection) (Fig. 1B) could not sufficiently stimulate the host immune system.
Not surprisingly, 4 to 8 weeks postinfection, the cytokine responses to WT and iscl−/+ISCL parasites were skewed toward Th2 in BALB/c mice (high in IL-4/IL-10, low in IFN-γ) and toward Th1 in C57BL/6 mice (high in IFN-γ, low in IL-4/IL-10) (Fig. 3). In contrast, iscl− parasites failed to induce the production of Th2 cytokines such as IL-4 or IL-10 in BALB/c mice or Th1 cytokines such as IFN-γ in C57BL/6 mice (Fig. 3). Consequently, these mutants did not trigger polarized T cell responses. Again, our results suggest that a low level of iscl− parasites cannot sufficiently engage the host immune system to produce a response that will either favor parasite survival (Th2) or lead to parasite elimination (Th1).
Overall, the cytokine response induced by iscl− mutants is very similar to what was observed in mice infected by another “persistence-without-pathology” mutant, the lpg2− mutant (28). Previous reports and our own study showed that lpg2− mutant-infected animals are protected from secondary, virulent WT L. major challenges (12, 28) (see Fig. S2 in the supplemental material). In general, vaccination with attenuated strains is considered safer than the “leishmanization” approach using virulent L. major (13, 14). Although the immunological mechanism for such protection is not entirely clear, it has been suspected that live, persistent parasites are required to elicit and maintain immunity (32). The similarity between iscl− and lpg2− mutants prompted us to examine whether mice infected with iscl− mutants were also protected from WT challenge. Surprisingly, iscl− parasites did not confer any protection in mice even when they were administered with CpG adjuvant (Fig. 4). Thus, having live, attenuated parasites is not sufficient for mice to develop immunity to L. major. The extremely low level of persistence of iscl− parasites (50 to 100 per mouse) may account for the lack of protection (lpg2− parasites may persist at 100 to 1,000 per mouse) (24). Alternatively, it is possible that the degradation of host sphingomyelin is required for the maintenance of anti-Leishmania effector memory T cells.
ISCL is essential for L. major growth in macrophages but not the ability to suppress NO/IL-12 production.Multiple classes of phagocytes are involved in the establishment of Leishmania infection. While neutrophils and dendritic cells are imperative for the initial parasite uptake and antigen presentation (16, 22), macrophages are considered the definitive host cells supporting Leishmania proliferation. Clearly, the ability to degrade SLs is pivotal for the survival and multiplication of L. major in murine macrophages (Fig. 5A and B) (35). A recent report shows that Leishmania deactivation of macrophages is mediated by the GP63-dependent cleavage of transcription factor AP-1 (4). Here we investigated whether ISCL was also involved in the alteration of host cell signaling. Previously, a 2- to 3-fold increase in ceramide production was observed in murine macrophages following L. donovani infection, which might suppress the release of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α), leading to reduced NO/ROI production in macrophages (7–9). It is unclear whether ISCL contributes to the increased ceramide production or whether such a mechanism applies to other Leishmania species. Our results show that L. major iscl− mutants were able to invade macrophages silently and suppress the production of NO/IL-12 (Fig. 5G and H). Therefore, the degradation of host sphingomyelin (and/or the production of ceramide from this pathway) is not required for the downregulation of macrophage activation. In addition, the inability of iscl− mutants to grow in iNOS- or phox-deficient macrophages suggests that the defect is not due to hypersensitivity to RNIs/ROIs. Instead, ISCL is likely involved in other aspects of intracellular growth for Leishmania.
ISCL is required for tolerance to acidic pH during stationary phase.During their life cycle, Leishmania promastigotes undergo a differentiation process from replicative, nonvirulent log phase (containing mostly procyclics) to nonreplicative, highly virulent stationary phase (enriched in metacyclics) (20, 21). Previously, we showed that iscl− mutants had no apparent defects in the transition from procyclics to metacyclics, except that more round cells were observed in late stationary phase (35). Here we tested whether stationary-phase promastigotes of iscl− parasites are fully adapted to the harsh conditions in macrophages. Interestingly, iscl− promastigotes were hypersensitive to acidic pH (pH 5.0) in stationary phase (Fig. 6) but not to other adverse factors, such as elevated temperature, ROIs, and RNIs (see Fig. S4 and S5 in the supplemental material). This defect may explain the poor survival of iscl− mutants in macrophages, where parasites are expected to establish infection in an acidic parasitophorous vacuole within 24 h (Fig. 5). Although somewhat delayed, phagosomal acidification still occurs in L. major-infected macrophages (Fig. S7) and may lead to increased death of iscl− mutants. In agreement with that, BAF treatment of macrophages (which blocked lysosomal acidification) significantly improved the viability of iscl− mutants during the first 24 h (Fig. 7; see also Fig. S6 and S7). It is therefore possible that BAF allows iscl− mutants to establish infection and differentiate into amastigotes within 24 h. It is not clear whether amastigote forms of iscl− mutants are sensitive to acidic pH, a possibility to be tested using Leishmania species that can form axenic amastigotes. Future studies will also determine whether ISCL possesses any additional roles besides conferring acid resistance during the establishment of infection.
The mechanism by which ISCL contributes to pH resistance is not clear. Notably, only stationary-phase (not log-phase) iscl− promastigotes were hypersensitive to acidic pH (Fig. 6; see also Fig. S3 in the supplemental material). Given its mitochondrial localization (in promastigotes), ISCL may play a role in mitochondrial function, which would explain the defect of iscl− mutants in stationary phase when sugars are depleted and cells have to depend on mitochondrial activity to produce energy (35) (Fig. 6). Such a defect could be exacerbated with acidic pH, under which condition more energy is needed to maintain the intracellular pH homeostasis via membrane-bound proton pumps.
In summary, our studies demonstrate that SL degradation is not essential for the downregulation of macrophage activation or suppression of the Th1 response. ISCL is also not required for resistance to elevated temperatures or the activities of ROIs/RNIs. However, SL degradation does play an important role in the tolerance to acidic pH. Because ISCL is both an SMase and an IPCase, further analyses are needed to determine which activity is required for acid resistance and whether it is sufficient to fully complement iscl− parasites' defects in mammals. In addition, future work will also focus on the localization and function of ISCL in the amastigote stage to elucidate the essential roles of this enzyme (besides conferring resistance to acidic pH) in Leishmania-host interactions. Understanding the contribution of ISCL in parasite virulence may aid the development of new selective inhibitors to control leishmaniasis.
ACKNOWLEDGMENTS
This work was supported by Public Health Service grants 1R56AI081781 (K.Z.), 5R03AI076662 (K.Z.), and 5R01AI043003 (L.S.) from the National Institute of Allergy and Infectious Diseases.
FOOTNOTES
- Received 10 January 2011.
- Returned for modification 2 February 2011.
- Accepted 9 May 2011.
- Accepted manuscript posted online 16 May 2011.
† Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00037-11.
- Copyright © 2011, American Society for Microbiology. All Rights Reserved.
