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 autophag...
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BMC
2015-07-01
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| Series: | Parasites & Vectors |
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| Online Access: | https://doi.org/10.1186/s13071-015-0974-3 |
| _version_ | 1797811835704967168 |
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| author | Benjamin Frank Ana Marcu Antonio Luis de Oliveira Almeida Petersen Heike Weber Christian Stigloher Jeremy C. Mottram Claus Juergen Scholz Uta Schurigt |
| author_facet | Benjamin Frank Ana Marcu Antonio Luis de Oliveira Almeida Petersen Heike Weber Christian Stigloher Jeremy C. Mottram Claus Juergen Scholz Uta Schurigt |
| author_sort | Benjamin Frank |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-13T07:29:40Z |
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| institution | Directory Open Access Journal |
| issn | 1756-3305 |
| language | English |
| last_indexed | 2024-03-13T07:29:40Z |
| publishDate | 2015-07-01 |
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| spelling | doaj.art-a6a6490b278f427c83cfd4c1e143bda72023-06-04T11:11:39ZengBMCParasites & Vectors1756-33052015-07-018113110.1186/s13071-015-0974-3Autophagic digestion of Leishmania major by host macrophages is associated with differential expression of BNIP3, CTSE, and the miRNAs miR-101c, miR-129, and miR-210Benjamin Frank0Ana Marcu1Antonio Luis de Oliveira Almeida Petersen2Heike Weber3Christian Stigloher4Jeremy C. Mottram5Claus Juergen Scholz6Uta Schurigt7Institute for Molecular Infection Biology, University of WuerzburgInstitute for Molecular Infection Biology, University of WuerzburgWellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, University of GlasgowInterdisciplinary Center for Clinical Research (IZKF), University of WuerzburgDivision of Electron Microscopy, Biocenter of the University of WuerzburgWellcome Trust Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, University of GlasgowInterdisciplinary Center for Clinical Research (IZKF), University of WuerzburgInstitute for Molecular Infection Biology, University of WuerzburgAbstract 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.https://doi.org/10.1186/s13071-015-0974-3AutophagyBNIP3CTSEElectron tomographyLeishmania majorMacrophages |
| spellingShingle | Benjamin Frank Ana Marcu Antonio Luis de Oliveira Almeida Petersen Heike Weber Christian Stigloher Jeremy C. Mottram Claus Juergen Scholz Uta Schurigt 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 Parasites & Vectors Autophagy BNIP3 CTSE Electron tomography Leishmania major Macrophages |
| 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 |
| title_full | 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 |
| title_fullStr | 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 |
| title_full_unstemmed | 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 |
| title_short | 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 |
| title_sort | 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 |
| topic | Autophagy BNIP3 CTSE Electron tomography Leishmania major Macrophages |
| url | https://doi.org/10.1186/s13071-015-0974-3 |
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