Activation of p38 Mitogen-Activated Protein Kinase Attenuates Leishmania donovani Infection in Macrophages

Reagents and antibodies.

RPMI 1640, M199, fetal calf serum (FCS), penicillin, and streptomycin were purchased from GIBCO/BRL. SB203580 was obtained from Calbiochem (Nottingham, United Kingdom). Anisomycin (Streptomyces griseolus) was obtained from Sigma. Antibodies against phosphorylated and nonphosphorylated p38 MAPK, horseradish peroxidase-conjugated anti-rabbit immunoglobulin G, and PD98059 were obtained from New England Biolabs (Hitchin, Hertfordshire, United Kingdom).

Parasites.

L. donovani (MHOM/ET/67/L82) amastigotes were periodically recovered from the spleens of infected hamsters (provided by V. Yardley and S. L. Croft, London School of Hygiene and Tropical Medicine). Promastigotes were derived through amastigote culture in M199 supplemented with 10% FCS, 2 mM glutamine, penicillin G (100 U/ml), streptomycin sulfate (100 μg/ml), 2 mM glutamate, 1 mM sodium pyruvate, and 10 mM HEPES at 26°C. Promastigotes were used at the stationary phase of growth, which was reached at approximately 7 to 9 days after subculture (39). Parasites were kept in culture by weekly passaging.

Cell preparation and culture.

Peripheral blood mononuclear cells were isolated from buffy coats obtained from healthy donors (National Blood Service, North London Branch, London, United Kingdom) on Histopaque (density, 1.077 g/ml; Sigma). The mononuclear cell fraction was washed three times and suspended in warm RPMI 1640 medium supplemented with 2 mM glutamine, penicillin G (100 U/ml), streptomycin sulfate (100 μg/ml), 2 mM glutamate, 1 mM sodium pyruvate, and 10 mM HEPES. Cell viability was determined using trypan blue (Sigma). Cells (2 × 10⁶) were dispensed into 24-well plates for the preparation of cell lysates, and cells (4 × 10⁵) were dispensed in eight-well chamber slides (Labtek) for infection assays and allowed to adhere for 2 h at 37°C in 5% CO_2. Nonadherent cells were washed off, and wells were replenished with supplemented RPMI 1640 containing 10% FCS for 5 to 6 days to generate a more differentiated phenotype.

Infection of macrophages.

Macrophages were either treated pre- or postinfection with a p38 MAPK inhibitor (SB203580), p42/44 MAPK (ERK1/2) inhibitor PD98059, or anisomycin at the indicated concentrations. Cells were then infected at a parasite-to-macrophage ratio of 20:1 for 2 h at 37°C in a humidified atmosphere of 5% CO₂, after which noningested promastigotes were washed with warm RPMI. Infected cells were fixed with methanol for 10 min, air dried, and stained with Giemsa's stain (BDH) diluted 1:9 in Sorenson's buffer (4.8 mM Na₂HPO₄, 1.9 mM KH₂PO₄)_. The numbers of infected macrophages and parasite burden (number of parasites per 100 infected macrophages) were determined by counting 500 macrophages on each duplicate of coverslips. Each experiment was repeated at least three times. Results were expressed as mean ± standard error of the mean (SEM) of individual experiments. In other experiments, infected macrophages were treated with anisomycin and/or LPS and supernatants were collected after 18 h for cytokine analyses.

Antileishmanial activity of anisomycin.

Metacyclic promastigotes or amastigotes were seeded into 96-well flat-bottom plates (Nunc) at 10⁶/well to a final volume of 100μl of M199-10% FCS, containing increasing amounts of anisomycin. After incubation for the given times at 26°C, the number of viable leishmaniae/well was determined by adding 10 μl of 5-mg/ml 3-(4,5-dimethylthiazil-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma) for 4 h, and the reaction was stopped with 100 μl of 50% isopropanol and 10% sodium dodecyl sulfate (SDS) for 30 min at room temperature. The relative optical density/well was determined by using an automatic microplate reader (Dynatech Labs) at 570 nm.

Preparation of cell extracts for Western blot analysis.

After treatment, cells (2 × 10⁵/sample) were washed twice with RPMI and lysed with ice-cold lysis buffer (50 mM Tris-HCl [pH 7.4], 1 mM EGTA, 150 mM NaCl, 1% [vol/vol] Triton X-100, 1 mM phenylmethylsulfonyl, aprotinin (10 μg/ml), 10 mM EDTA, 1 mM NaF, 1 mM Na₃VO₄). Lysates were centrifuged at 13,000 × g at 4°C for 10 min, and the resulting supernatants were transferred to fresh tubes and stored at −40°C until required.

Western blot analysis.

Cell lysates were treated with an equal volume of 2× SDS sample buffer (62.5 mM Tris-HCl [pH 6.8], 2% [wt/vol] SDS, 10% [vol/vol] glycerol, 50 mM dithiothreitol, 0.1% [wt/vol] bromphenol blue) and then electrophoresed on a 10% (acrylamide:bisacrylamide, 40:1 [wt/vol]). Proteins were transferred to a polyvinylidene difluoride membrane using a semidry apparatus under standard conditions. The membrane was then incubated successively with and 20 mM Tris (pH 7.6)-0.137 M NaCl-0.1% (vol/vol) Tween-20 (TBST) containing 0.1% (wt/vol) milk powder at room temperature for 1 h, with rabbit antibodies specific for phosphorylated or nonphosphorylated p38 MAPK overnight at 4°C, and then with horseradish peroxidase-labeled anti-rabbit antibody for 1 h. After each incubation, the membrane was extensively washed with TBST, and the immunoreactive band was detected with enhanced-chemiluminescence-detecting reagents and developed with Kodak MR film.

Cytokine measurement.

TNF-α was measured by enzyme-linked immunosorbent assay using TNF-α monoclonal antibody (2 μg/ml; Pharmingen) as the capture antibody and biotinylated anti-human TNF-α monoclonal antibody (1 μg/ml; Pharmingen) as the detecting antibody. The development was made with streptavidin-peroxidase conjugate (DAKO) and 3,3′,5,5′-tetramethylbenzinedihydro-chloride tablets (Sigma) as the substrate. Plates were read at 450 nm, and TNF-α concentration was calculated by reference to a standard curve of human TNF-α (NIBSC, South Mimms, United Kingdom).

Statistical analysis.

Except where otherwise stated, the results shown are representative data from a minimum of three similar experiments, which yielded comparable results. Differences between experimental groups were examined using Student's t test for independent means (Fig P for Windows).

Inhibition of p38 MAPK augments L. donovani infection.

To examine the role of p38 MAPK in leishmania infection, the effect of a selective p38 MAPK inhibitor, SB203580, was assessed. SB203580 is a pyrindyl imidazole which binds with a high affinity to p38 near the ATP binding site, thus rendering p38 MAPK inactive (8). Macrophages were treated with increasing concentrations of SB203580 prior to a 2-h infection with L. donovani promastigotes. Compared to the control, p38 MAPK inhibition induced a significant increase in the subsequent successful infection by L. donovani promastigotes as shown by an increase in the percentage of macrophages infected and parasite burden (number of parasites per 100 infected macrophages) (Fig. 1). When combined, this represented an approximately 50% increase in parasite numbers per macrophage. These concentrations of SB203580 did not induce any toxicity as determined by microscopic observations and trypan blue exclusion (data not shown). Macrophages first infected for 2 h and then subsequently treated with SB203580 for an hour resulted in a slight increment in the percentage of macrophages infected (P < 0.05; 0.01μg/ml) with no significant effect on parasite burden (data not shown).

Considering that SB203580 enhances parasite invasion, it is possible that leishmania parasites may not completely inhibit this kinase during infection. We therefore determined whether p38 MAPK is altered during infection by measuring kinetics of its phosphorylation on exposure to L. donovani promastigotes. Compared to noninfected controls, L. donovani promastigotes induced a slight increase in p38 MAPK phosphorylation at 30 min and 2 h, with phosphorylation back to baseline by 18 h of infection (Fig. 2).

Anisomycin induces parasite inhibition in infected macrophages.

Following the observation that inhibition of p38 MAPK enhances L. donovani infection, we investigated the relevance of activating p38 MAPK on L. donovani infection using anisomycin. Anisomycin acts as a signaling agonist at concentrations at or below 100 ng/ml to selectively activate stress-related MAPKs (p38 MAPK and JNK) (25, 41) with no toxicity. Macrophage monolayers stimulated with anisomycin for 1 h prior to infection with L. donovani promastigotes showed a significant decrease in both the percentages of macrophages infected and parasite burden (Fig. 3). When combined, this represented an approximately 50% reduction in intracellular parasites. This may have been as a result of inhibition of phagocytosis and hence a reduction in parasite uptake. Therefore, in order to examine survival without potential effects on uptake of parasites, L. donovani-infected macrophages were treated with increasing concentrations of anisomycin. Following treatment the percentage of macrophages infected was reduced, although there was no significant change in parasite burden within infected macrophages (Fig. 4). Lack of change in parasite burden could be as a result of anisomycin targeting different populations of infected macrophages. On further observation of such treated macrophages, only those macrophages containing few organisms (one to three parasites) responded to anisomycin treatment (Fig. 5). There was no change in anisomycin-treated macrophages with a higher parasite burden (more than six) compared to the control.

The antileishmanial effects observed may arise from a direct action of anisomycin on the parasites or by an effect of anisomycin on macrophages activated to eliminate parasites. We therefore assessed if anisomycin had any effect on amastigotes, the intracellular stage of Leishmania. Hamster-derived amastigotes were cultured in the presence of anisomycin at concentrations of up to 100 μg/ml at 37°C. After a period of up to 24 h, parasite viability was assayed by the ability of viable parasites to reduce MTT to formazan. Anisomycin had no effect on the viability of amastigotes at concentrations up to 10 μg/ml, suggesting that the observed inhibition was not due to a direct effect on the parasites. Similarly, treatment of the extracellular infective promastigote form with anisomycin at 26°C over a period of 72 h did not have any effect on the promastigote's growth and viability (data not shown).

Anisomycin does not reverse the effect of SB203580 on Leishmania infection.

Anisomycin has been shown to stimulate both the JNK/SAPK and p38 MAPK cascades. Hence, the effects of anisomycin seen so far could be a result of activation of either one or both p38 MAPK and JNK cascades. It was therefore of interest to investigate whether specific p38 MAPK inhibition was able to reverse the effects of anisomycin on Leishmania infection. Macrophages formerly treated with increasing concentrations of SB203580 were infected with L. donovani for 2 h. Uningested parasites were washed off, and infected macrophage monolayers were treated with anisomycin (100 ng/ml). As previously observed, anisomycin reduced the percentage of macrophages compared to control macrophages. Surprisingly, the presence of SB203580 completely prevented this reduction (Fig. 6), indicating the significance of p38 MAPK in the anisomycin-induced killing of L. donovani.

Effect of ERK inhibitor on L. donovani infection.

To examine whether ERK was involved in L. donovani infection, macrophages were preincubated with PD98059 1 h prior to infection. PD98059 is a selective and potent inhibitor of ERK and mediates its effects by binding to and inactivating the ERK-specific MAPK kinase, MEK (2, 10). It has no effects on any of the components of JNK and p38 MAPK cascades (30). Inhibition of ERK showed an increase in the percentage of macrophages (1.0 μM, P < 0.05) (Fig. 7). This increase was not as pronounced as that seen with SB203580. Furthermore, the presence of PD98059 did not inhibit anisomycin-induced parasite inhibition. There was no significant change in the parasite burden (data not shown).

Anisomycin-induced killing does not increase with time.

Anisomycin induced killing within 2 h; therefore, we tested whether this effect was altered over longer periods of time. Macrophages infected with L. donovani promastigotes for 18 h and treated with anisomycin (100 ng/ml) for 1, 6, 12, 24, 48, and 72 h resulted in 93.7% ± 2.0% (P = 0.08), 57.2% ± 8.7% (P < 0.05), 44.1% ± 2.6% (P < 0.02), 49.6% ± 4.0% (P < 0.02), 51.3% ± 1.0% (P < 0.001), and 48.2% ± 6.8% (P < 0.02) inhibition of percentage of macrophages infected, respectively, compared to control. These results indicate that the effects of anisomycin were not reduced by longer infection periods of 18 h, nor was a further killing observed beyond 6 h and up to 48 h.

LPS does not enhance anisomycin-induced inhibition.

IFN-γ and LPS cooperatively enhance killing of intracellular Leishmania in macrophages, an action that is dependent on TNF-α and NO secretion (14-16). It has been well established that among the MAPKs, p38 MAPK plays an important role in LPS-induced TNF-α production in many different cell types (19, 20) We therefore assessed whether LPS would synergize with anisomycin and cooperatively enhance killing of intracellular Leishmania. Macrophages (18 h infected) were treated with various concentrations of anisomycin and LPS. As shown in Fig. 8A, the presence of LPS did not further enhance anisomycin-induced parasite inhibition. Infection of macrophages with L. donovani has been shown to interfere with cytokine production (9). Anisomycin, unlike LPS was unable to induce the production of TNF-α (Fig. 8B). The presence of anisomycin moderately enhanced LPS-induced TNF-α production. In addition, TNF-α production from these cells treated with a combination of anisomycin and LPS confirmed that anisomycin was not toxic to the cells at the concentrations used.

Anisomycin phosphorylates p38 MAPK in infected macrophages.

As we have previously observed, SB203580 inhibited anisomycin-induced parasite inhibition (Fig. 6), indicating that activation of p38 MAPK was involved in anisomycin-induced parasite inhibition. We therefore used Western blotting to examine the activation of p38 MAPK by anisomycin in L. donovani-infected macrophages. Positive controls consisted of naïve uninfected macrophages stimulated with anisomycin for the same times as infected macrophages. Activation of p38 MAPK requires phosphorylation at threonine and tyrosine residues. Immunoblot analyses using anti-phospho-specific p38 antibodies were performed. As shown in Fig. 8A and C, naïve macrophages treated with 100-ng/ml anisomycin had a maximal activation of p38 MAPK between 15 min and 2 h. Whereas anisomycin induced a strong and transient phosphorylation of p38 MAPK in naïve macrophages, there was a superinduction of phosphorylated p38 MAPK in L. donovani-infected macrophages (Fig. 9B and C).