Combination of paromomycin and miltefosine promotes TLR4-dependent induction of antileishmanial immune response in vitro.

OBJECTIVES To evaluate the in vitro activity of antileishmanial drugs, paromomycin and miltefosine, to generate Th-1-biased immunomodulation in hosts against intracellular Leishmania donovani. METHODS In silico protein-ligand interaction and in vitro drug-cell interaction assays were performed. Interaction assays of TLR4-deficient HEK293 cells and HEK293 cells engineered to express either TLR4 or TLR2 with different concentrations of miltefosine and/or paromomycin sulphate were performed for 48 h. Differentially transfected human peripheral blood monocyte-derived macrophages (PBMFs) were treated with the drugs, and nuclear factor (NF)-κB promoter activity was measured using a κB-luciferase reporter construct. PBMFs were infected with L. donovani. Cultures were incubated with miltefosine or paromomycin sulphate over different concentrations, as mono-treatment or combined. The in vitro antileishmanial effect of the drugs on macrophage-bound L. donovani amastigotes was measured in terms of parasite killing and production of tumour necrosis factor-α (TNF-α) and nitric oxide. RESULTS Computational studies reveal that paromomycin and miltefosine interact with TLR4. Both drugs, as monotherapy or in combination, induce release of TNF-α and nitric oxide in a TLR4-dependent manner. Interestingly, the TLR4-dependent action of the drugs leads to NF-κB promoter activation through MyD88. In vitro, both the drugs kill macrophage-bound L. donovani by inducing release of TNF-α and nitric oxide in a TLR4-dependent manner. CONCLUSIONS The in vitro activity of paromomycin and miltefosine against host cells is TLR4 dependent. This has implications for: (i) evaluation of in vitro activity of combinational antileishmanial therapy; (ii) the evaluation of drug susceptibility of clinical isolates; and (iii) the standardization of in vitro antileishmanial assays for determining toxicity in hosts.