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

BackgroundAutophagy 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.MethodsBMDM 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.ResultsThe 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.ConclusionsAutophagy 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0974-3) contains supplementary material, which is available to authorized users.

Countervailing, time-dependent effects on host autophagy promote intracellular survival of Leishmania

“…For example, induction of autophagy correlated with increased parasite loads of L. amazonensis in BALB/c, but not in C57BL/6 mice (14). A recent study using L. major infected bone marrow derived macrophages showed host autophagy was detrimental to pathogen survival (17). In contrast, it was recently reported that a stationary phase, L. major inoculum containing significant number of apoptoticlike parasites activated human macrophages autophagy machinery, thereby dampening Tcell responses and promoting parasite survival (16).…”

“…Several species of leishmania have been observed to induce host autophagy, but the molecular mechanism(s) involved in the autophagy response have only recently begun to emerge (12)(13)(14)(15)(16)(17)(18)(19). For example, two recent studies reported a link between leishmania induced autophagy and a differentially expressed set of host micro RNAs (miRNAs), including MIR-30A-3p in infected cells (17,18).…”

“…For example, two recent studies reported a link between leishmania induced autophagy and a differentially expressed set of host micro RNAs (miRNAs), including MIR-30A-3p in infected cells (17,18).…”

“…The molecular mechanism(s) involved in leishmania mediated regulation of host autophagy have begun to emerge. For example, recent evidence suggests that select host microRNAs (miRNAs) may participate in regulation of host autophagy in response to leishmania infection (17,18). It has also been shown that autophagy induction through endosomal Toll-like receptors play a role in macrophages conferring resistance against Leishmania major infections.…”

Leishmania (Leishmania) amazonensis induces macrophage miR-294 and miR-721 expression and modulates infection by targeting NOS2 and L-arginine metabolism

“…Recent studies demonstrated that L. major and L. donovani infection induce alteration of human and murine host miRNA profiles2526272829. Here, we investigate the role of L .…”