Miltefosine efficiently eliminates Leishmania major amastigotes from infected murine dendritic cells without altering their immune functions

Abstract

As a treatment for leishmaniasis, miltefosine exerts direct toxic effects on the parasites. Miltefosine also modulates immune cells such as macrophages, leading to parasite elimination via oxidative radicals. Dendritic cells (DC) are critical for initiation of protective immunity against Leishmania through induction of Th1 immunity via interleukin 12 (IL-12). Here, we investigated the effects of miltefosine on DC in Leishmania major infections. When cocultured with miltefosine for 4 days, the majority of in vitro-infected DC were free of parasites. Miltefosine treatment did not influence DC maturation (upregulation of major histocompatibility complex II [MHC II] or costimulatory molecules, e.g., CD40, CD54, and CD86) or significantly alter cytokine release (IL-12, tumor necrosis factor alpha [TNF-alpha], or IL-10). Further, miltefosine DC treatment did not alter antigen presentation, since unrestricted antigen-specific proliferation of CD4+ and CD8+ T cells was observed upon stimulation with miltefosine-treated, infected DC. In addition, miltefosine application in vivo did not lead to maturation/emigration of skin DC. DC NO- production, a mechanism used by phagocytes to rid themselves of intracellular parasites, was also unaltered upon miltefosine treatment. Our data confirm prior studies indicating that in contrast to, e.g., pentavalent antimonials, miltefosine functions independently of the immune system, mostly through direct toxicity against the Leishmania parasite.

FIG. 1.

Survival of DC after treatment with miltefosine. DC were generated as immature cells from bone marrow of C57BL/6 mice and incubated with various doses of miltefosine (0 to 500 μM) at 1 × 10⁵ cells/100 μl. (A) After 48 h, the percentage of surviving DC was determined using propidium iodide (PI) exclusion via flow cytometry (20 to 100 ng/ml). Data are means plus standard errors of the means (SEM) (n ≥ 3; * and ***, P < 0.05 and P < 0.002 compared to untreated cells after 48 h). (B) Early apoptosis was determined by staining of miltefosine-treated DC with PI and annexin V after 18 and 72 h using flow cytometry. (C) DC were incubated with 5 μM miltefosine, and cell survival was monitored over the following 4 days. Data are percent PI⁻ cells relative to untreated controls at each time point ( mean ± SEM; n ≥ 2; ***, P < 0.002).

FIG. 2.

Miltefosine treatment abrogates L. major-induced upregulation of activation markers. (A and B) Bone marrow DC were incubated with 5, 25, or 50 μM miltefosine or 100 ng/ml LPS for 18 h (1 × 10⁵ cells/100 μl). Cell activation by miltefosine or LPS was assessed by determination of surface expression levels of MHC class II and the costimulatory molecules CD40, CD54, and CD86 (solid line, isotype staining; filled curve, specific-MAb staining). (C) Prior to miltefosine or LPS treatment, DC were infected overnight with amastigotes of L. major (5 parasites/cell; mean infection rate, 23.6% ± 1.4%) and washed. Subsequently, DC were stimulated with miltefosine or LPS as described above. Data are percent change in surface expression relative to untreated controls (mean ± SEM; n ≥ 3; *, P < 0.05; **, P < 0.005; ***, P < 0.002).

FIG. 3.

In vivo application of miltefosine does not lead to maturation of resident DC. (A) LPS, miltefosine, or PBS was injected intradermally as indicated. After 48 h, MHC II⁺ CD11c⁺ DC were enumerated by fluorescence-activated cell sorting (FACS) analysis. Representative dot blots from one of two experiments are shown. (B) Pooled data. The percentages of cells in the monocyte gate were calculated (mean ± SEM; n = 2).

FIG. 4.

Incubation with miltefosine does not significantly alter DC cytokine production. Untreated (A) or L. major-infected (B) DC were incubated with miltefosine or LPS at 1 × 10⁵ cells/100 μl. Cytokine levels were determined in supernatants harvested after 18 h using ELISA specific for TNF-α, IL-12p40, IL-12p70, and IL-10. Data are means plus SEM (n ≥ 3; *, P < 0.05).

FIG. 5.

L. major amastigotes are eliminated from infected DC by miltefosine treatment despite absent NO production. C57BL/6 DC were infected overnight with amastigotes at a cell/parasite ratio of 1:5 (A and B) or 1:10 (C). Some cells were left untreated. (A and B) Infected cells were washed thoroughly to remove extracellular parasites and plated at 1 × 10⁵ cells/100 μl. Half of the wells were additionally incubated with 50 μM miltefosine. Every 24 h, cells were harvested and subjected to centrifugation in a Cytospin, and the percentage of infected cells (A) as well as the number of intracellular parasites (B) was determined by light microscopy. Data are means plus SEM (n ≥ 3; ***, P < 0.002; *, P < 0.05). (C) After washing, cells were plated at 2 × 10⁶ cells/ml and incubated with 50 μM miltefosine or LPS (100 ng/ml) and IFN-γ (1,000 U/ml). Nitrite production was assessed after 72 h using the Griess reagent (means plus SEM; n = 7; LPS/IFN-γ, n = 4; *, P < 0.05).

FIG. 6.

Unaltered antigen presentation by miltefosine-treated, L. major-infected DC. CD4⁺ (A) and CD8⁺ (B) T cells were enriched from C57BL/6 mice that had been infected with L. major at least 6 weeks earlier. DC were incubated with L. major overnight or left untreated, washed, and incubated for 72 h in the presence or absence of miltefosine (50 μM). T cells (1 × 10⁵) were incubated with various concentrations of irradiated DC (starting with a T-cell/DC ratio of 10:1). After 48 h, cells were pulsed with 1 μCi [³H]thymidine for the last 18 h of incubation. Thymidine incorporation was determined using a liquid scintillation counter. Data are means plus SEM from ≥3 independent experiments.