Abstract
Autophagy plays important roles in the host immune response against mycobacterial infection. Mycobacterium tuberculosis (M. tuberculosis) can live in macrophages owing to its ability to evade attacks by regulating autophagic response. MicroRNAs (miRNAs) are small noncoding, endogenously encoded RNA which plays critical roles in precise regulation of macrophage functions. Whether miRNAs specifically influence the activation of macrophage autophagy during M. tuberculosis infection are largely unknown. In this study, we demonstrate that BCG infection of macrophages resulted in enhanced expression of miRNA-20a, which inhibits autophagic process by targeting ATG7 and ATG16L1 and promotes BCG survival in macrophages. Forced overexpression of miR-20a decreased the expression levels of LC3-II and the number of LC3 puncta in macrophages, and promoted BCG survival in macrophages, while transfection with miR-20a inhibitor had the opposite effect. Moreover, the inhibitory effect of miR-20a on autophagy was further confirmed by transmission electron microscopy (TEM) analysis. Quantification of autophagosomes per cellular cross-section revealed a significant reduction upon transfection with miR-20a mimic, but transfection with miR-20a inhibitor increased the number of autophagosomes per cellular cross-section. Moreover, silencing of ATG7 significantly inhibited autophagic response, and transfection with ATG7 siRNA plus miR-20a mimic could further decrease autophagic response. Collectively, our data reveal that miR-20a inhibits autophagic response and promotes BCG survival in macrophages by targeting ATG7 and ATG16L1, which may have implications for a better understanding of pathogenesis of M. tuberculosis infection.
Highlights
Autophagy is an evolutionarily conserved mechanism responsible for intracellular degradation of proteins and whole organelles and for resistance to pathogenic infection (Yang and Klionsky, 2010; Deretic et al, 2013)
In order to determine whether miR-20a contribute to Bacillus Calmette-Guérin (BCG) survival in macrophages by inhibiting autophagy, Quantitative real-time PCR (qRT-PCR) was used to determine the bacterial load in different treatment groups. qPCR assay showed that transfection with miR-20a mimic or ATG7 siRNA significantly increased the bacterial load of intracellular BCG in RAW264.7 cells, whereas transfection with miR-20a inhibitor significantly decreased the bacterial load of intracellular BCG (Figure 6)
Precise regulation of macrophages is crucial for maintaining mycobacterial latent infection and M. tuberculosis clearance
Summary
Autophagy is an evolutionarily conserved mechanism responsible for intracellular degradation of proteins and whole organelles and for resistance to pathogenic infection (Yang and Klionsky, 2010; Deretic et al, 2013). The autophagic process involves the formation of autophagosomes which are double membrane vesicles. Autophagosomes engulf portions of cytoplasm together with proteins, whole organelles or pathogens such as Mycobacterium tuberculosis (M. tuberculosis), become mature gradually along the endocytic pathway, acidify, and form autolysosomes by fusing with lysosome, and bring about degradation of inclusions in autophagic vacuoles. M. tuberculosis “enhanced intracellular survival” (eis) gene can inhibit host innate immune defenses by modulating autophagy, inflammation, and cell death through redox-dependent signaling (Shin et al, 2010). ATG7 participates in two important functions involved in autophagosome formation and in vesicle progression. ATG16L1 controls the elongation of the nascent autophagosomal membrane (Levine et al, 2011)
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