Miltefosine [hexadecylphosphocholine (HPC)] is the only orally bioavailable drug for the disease visceral leishmaniasis, which is caused by the protozoan parasite Leishmania donovani. Although miltefosine has direct leishmanicidal effects, evidence is mounting for its immune system-dependent effects. The mechanism of such indirect antileishmanial effects of miltefosine remains to be discovered. As platelet-activating factor and HPC share structural semblances and both induce killing of

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... ular Leishmania, we surmised that platelet-activating factor (PAF) receptor had a significant role in the antileishmanial function of miltefosine. The proposition was supported by molecular dynamic simulation of HPC docking into PAF receptor and by comparison of its leishmanicidal function on PAF receptor-deficient macrophages and mice under HPC treatment. We observed that compared with wild-type macrophages, the PAF receptor-deficient macrophages showed 1) reduced binding of a fluorescent analog of HPC, 2) decreased TNF-a production, and 3) lower miltefosine-induced killing of L. donovani. Miltefosine exhibited significantly compromised leishmanicidal function in PAF receptor-deficient mice. An anti-PAF receptor Ab led to a significant decrease in miltefosine-induced intracellular Leishmania killing and IFN-g production in a macrophage-T cell coculture system. These results indicate significant roles for PAF receptor in the leishmanicidal activity of HPC. The findings open new avenues for a more rational understanding of the mechanism of action of this drug as well as for improved therapeutic strategies. FIGURE 2. Modulation of antileishmanial activity of macrophages by PAF receptor. (A) BALB/c-derived thioglycolate-elicited macrophages were infected with L. donovani promastigotes for 48 h, followed by treatment with miltefosine (HPC) in the presence of the indicated doses of the anti-PAF receptor Ab. An isotype-matched Ab did not have any effect on the antileishmanial effect of HPC (data not plotted). *p , 0.001. (B) TNF-a production, as assessed by ELISA, from the BALB/c-derived thioglycolate-elicited macrophages 24 h after miltefosine treatment was significantly inhibited by the anti-PAF receptor Ab in a dose-dependent manner. **p , 0.001. (C) HPC-induced TNF-a production by the thioglycolate-elicited WT (BALB/c) and PAF receptor-deficient macrophages. Thioglycolate-elicited macrophages were infected with L. donovani promastigotes for 4 h. The UIM and infected IM were treated with miltefosine (HPC) for 24 h. (D) BALB/c-derived peritoneal macrophages were infected with L. donovani promastigotes for 48 h, followed by treatment with anti-PAF receptor or anti-TNF-a Ab for 24 h. Following the treatment, the parasite loads in macrophages were calculated and expressed as indicated. Each of these experiments was performed three times. Results from one representative experiment are shown in this figure. Bars stand for means 6 SD. FIGURE 5 . Antileishmanial activity of miltefosine is compromised in PAF receptor-deficient macrophages. Thioglycolate-elicited peritoneal macrophages from BALB/c and PAF receptor-deficient (PAF receptor-KO) mice were obtained 4-5 d after thioglycolate injection. Macrophages were infected with L. donovani promastigotes at a macrophage/parasite ratio of 1:10. The nonphagocytized parasites were washed with PBS, and some cultures were treated with the indicated concentrations of HPC for 4, 24, or 72 h. The cultures were fixed and stained with Giemsa stain, and the percentage of IM and number of amastigotes per IM were enumerated. The cultures were in triplicates. Statistical analysis was performed by two-way ANOVA followed by the Tukey test. The results are representative of three independent experiments performed with similar results. *p , 0.05.