title: Combination of paromomycin and miltefosine promotes TLR4-dependent induction of antileishmanial immune response in vitro.
abstract: OBJECTIVE
  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.
authors: Departments of Molecular Parasitology, Bioinformatics and Clinical Medicine, Rajendra Memorial Research Institute of Medical Sciences, Indian Council of Medical Research (ICMR), Agamkuan, Patna, India.
authors: Sushmita
authors: S
authors: Das
authors: Mukta
authors: M
authors: Rani
authors: Krishna
authors: K
authors: Pandey
authors: Ganesh Chandra
authors: GC
authors: Sahoo
authors: Vidya Nand
authors: VN
authors: Rabidas
authors: Dharmendra
authors: D
authors: Singh
authors: Pradeep
authors: P
authors: Das
mesh_terms: D000293:Adolescent; D000328:Adult; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D019677:Artificial Gene Fusion; D002470:Cell Survival; D002478:Cells, Cultured; D005260:Female; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D006801:Humans; D007155:Immunologic Factors / Q000494:pharmacology*; D007893:Leishmania donovani / Q000276:immunology*; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D008264:Macrophages / Q000187:drug effects / Q000276:immunology / Q000469:parasitology; D008297:Male; D016328:NF-kappa B / Q000096:biosynthesis; D009569:Nitric Oxide / Q000378:metabolism; D010303:Paromomycin / Q000494:pharmacology*; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology; D051197:Toll-Like Receptor 4 / Q000819:agonists*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D055815:Young Adult
chemical_list: D000981:Antiprotozoal Agents; D007155:Immunologic Factors; D016328:NF-kappa B; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D010767:Phosphorylcholine; D009569:Nitric Oxide; C039128:miltefosine; D010303:Paromomycin; D008156:Luciferases