title: Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210.
abstract: BACKGROUND
  Autophagy participates in innate immunity by eliminating intracellular pathogens. Consequently, numerous microorganisms have developed strategies to impair the autophagic machinery in phagocytes. In the current study, interactions between Leishmania major (L. m.) and the autophagic machinery of bone marrow-derived macrophages (BMDM) were analyzed.
  
  
  METHODS
  BMDM were generated from BALB/c mice, and the cells were infected with L. m. promastigotes. Transmission electron microscopy (TEM) and electron tomography were used to investigate the ultrastructure of BMDM and the intracellular parasites. Affymetrix chip analyses were conducted to identify autophagy-related messenger RNAs (mRNAs) and microRNAs (miRNAs). The protein expression levels of autophagy related 5 (ATG5), BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), cathepsin E (CTSE), mechanistic target of rapamycin (MTOR), microtubule-associated proteins 1A/1B light chain 3B (LC3B), and ubiquitin (UB) were investigated through western blot analyses. BMDM were transfected with specific small interfering RNAs (siRNAs) against autophagy-related genes and with mimics or inhibitors of autophagy-associated miRNAs. The infection rates of BMDM were determined by light microscopy after a parasite-specific staining.
  
  
  RESULTS
  The experiments demonstrated autophagy induction in BMDM after in vitro infection with L. m.. The results suggested a putative MTOR phosphorylation-dependent counteracting mechanism in the early infection phase and indicated that intracellular amastigotes were cleared by autophagy in BMDM in the late infection phase. Transcriptomic analyses and specific downregulation of protein expression with siRNAs suggested there is an association between the infection-specific over expression of BNIP3, as well as CTSE, and the autophagic activity of BMDM. Transfection with mimics of mmu-miR-101c and mmu-miR-129-5p, as well as with an inhibitor of mmu-miR-210-5p, demonstrated direct effects of the respective miRNAs on parasite clearance in L. m.-infected BMDM. Furthermore, Affymetrix chip analyses revealed a complex autophagy-related RNA network consisting of differentially expressed mRNAs and miRNAs in BMDM, which indicates high glycolytic and inflammatory activity in the host macrophages.
  
  
  CONCLUSIONS
  Autophagy in L. m.-infected host macrophages is a highly regulated cellular process at both the RNA level and the protein level. Autophagy has the potential to clear parasites from the host. The results obtained from experiments with murine host macrophages could be translated in the future to develop innovative and therapeutic antileishmanial strategies for human patients.
authors.affiliation: Institute for Molecular Infection Biology, University of Wuerzburg, Josef-Schneider-Str. 2/D15, 97080, Wuerzburg, Germany. benjamin.frank@stud-mail.uni-wuerzburg.de.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Frank
authors.affiliation: Institute for Molecular Infection Biology, University of Wuerzburg, Josef-Schneider-Str. 2/D15, 97080, Wuerzburg, Germany. ana.marcu@stud-mail.uni-wuerzburg.de.
authors.forename: Ana
authors.initials: A
authors.lastname: Marcu
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, G12 8TA, UK. petersen.swe@gmail.com.|Laboratório de Patologia e Biointervenção, Fundação Oswaldo Cruz-BA, Salvador, Bahia, Brazil. petersen.swe@gmail.com.
authors.forename: Antonio Luis
authors.initials: AL
authors.lastname: de Oliveira Almeida Petersen
authors.affiliation: Interdisciplinary Center for Clinical Research (IZKF), University of Wuerzburg, Wuerzburg, Germany. weber_h2@ukw.de.
authors.forename: Heike
authors.initials: H
authors.lastname: Weber
authors.affiliation: Division of Electron Microscopy, Biocenter of the University of Wuerzburg, Wuerzburg, Germany. christian.stigloher@uni-wuerzburg.de.
authors.forename: Christian
authors.initials: C
authors.lastname: Stigloher
authors.affiliation: Wellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, G12 8TA, UK. jeremy.mottram@glasgow.ac.uk.
authors.forename: Jeremy C
authors.initials: JC
authors.lastname: Mottram
authors.affiliation: Interdisciplinary Center for Clinical Research (IZKF), University of Wuerzburg, Wuerzburg, Germany. scholz_c@ukw.de.
authors.forename: Claus Juergen
authors.initials: CJ
authors.lastname: Scholz
authors.affiliation: Institute for Molecular Infection Biology, University of Wuerzburg, Josef-Schneider-Str. 2/D15, 97080, Wuerzburg, Germany. uta.schurigt@gmx.de.
authors.forename: Uta
authors.initials: U
authors.lastname: Schurigt
mesh_terms: D000818:Animals; D001343:Autophagy / Q000502:physiology*; D020059:Cathepsin E / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000276:immunology*; D018320:Leishmania major / Q000276:immunology*; D008264:Macrophages / Q000502:physiology; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C508686:BNip3 protein, mouse; D008565:Membrane Proteins; D035683:MicroRNAs; D024101:Mitochondrial Proteins; D020059:Cathepsin E; C533926:Ctse protein, mouse