title: TLR9 and MyD88 are crucial for the maturation and activation of dendritic cells by paromomycin-miltefosine combination therapy in visceral leishmaniasis.
abstract: OBJECTIVE
  The combination of paromomycin-miltefosine is a successful anti-leishmanial therapy in visceral leishmaniasis (VL). This encouraged us to study its effect on Toll-like receptor (TLR)-mediated immunomodulation of dendritic cells (DC), as DC maturation and activation is crucial for anti-leishmanial activity.
  
  
  METHODS
  In silico protein-ligand interaction and biophysical characterization of TLR9-drug interaction was performed. Interaction assays of HEK293 cells with different concentrations of miltefosine and/or paromomycin were performed, and NF-κB promoter activity measured. The role of TLR9 and MyD88 in paromomycin/miltefosine-induced maturation and activation of DCs was evaluated through RNA interference techniques. The effect of drugs on DCs was measured in terms of counter-regulatory production of IL-12 over IL-10, and characterized by chromatin immunoprecipitation assay at the molecular level.
  
  
  RESULTS
  Computational and biophysical studies revealed that paromomycin/miltefosine interact with TLR9. Both drugs, as a monotherapy/combination, induced TLR9-dependent NF-κB promoter activity through MyD88. Moreover, the drug combination induced TLR9/MyD88-dependent functional maturation of DCs, evident as an up-regulation of co-stimulatory markers, enhanced antigen presentation by increasing MHC II expression, and increased stimulation of naive T-cells to produce IFN-γ. Both drugs, by modifying histone H3 at the promoter level, increased the release of IL-12, but down-regulated IL-10 in a TLR9-dependent manner.
  
  
  CONCLUSIONS
  These results provide the first evidence that the combination of paromomycin-miltefosine critically modifies the maturation, activation and development of host DCs through a mechanism dependent on TLR9 and MyD88. This has implications for evaluating the success of other combination anti-leishmanial therapies that act by targeting host DCs.
authors.affiliation: Department of Molecular Parasitology, Rajendra Memorial Research Institute of Medical Sciences, Indian Council of Medical Research, Bihar, India.
authors.forename: Sushmita
authors.initials: S
authors.lastname: Das
authors.forename: Mukta
authors.initials: M
authors.lastname: Rani
authors.forename: Vidyanand
authors.initials: V
authors.lastname: Rabidas
authors.forename: Krishna
authors.initials: K
authors.lastname: Pandey
authors.forename: Ganesh Chandra
authors.initials: GC
authors.lastname: Sahoo
authors.forename: Pradeep
authors.initials: P
authors.lastname: Das
mesh_terms: D000328:Adult; D000981:Antiprotozoal Agents / Q000494:pharmacology* / Q000627:therapeutic use; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000166:cytology / Q000187:drug effects* / Q000382:microbiology; D004338:Drug Combinations; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D016753:Interleukin-10 / Q000235:genetics / Q000378:metabolism; D018664:Interleukin-12 / Q000235:genetics / Q000378:metabolism; D007898:Leishmaniasis, Visceral / Q000188:drug therapy / Q000378:metabolism*; D008297:Male; D053594:Myeloid Differentiation Factor 88 / Q000378:metabolism*; D010303:Paromomycin / Q000494:pharmacology* / Q000627:therapeutic use; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000494:pharmacology / Q000627:therapeutic use; D051217:Toll-Like Receptor 9 / Q000378:metabolism*
chemical_list: D000981:Antiprotozoal Agents; D004338:Drug Combinations; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; C495371:TLR9 protein, human; D051217:Toll-Like Receptor 9; D010767:Phosphorylcholine; D016753:Interleukin-10; D018664:Interleukin-12; C039128:miltefosine; D010303:Paromomycin
keywords: DC maturation; TLR9; combination therapy; miltefosine; paromomycin; visceral leishmaniasis