Abstract
Autophagy is a lysosomal self-digestion pathway that maintains internal homeostasis inside cells and critical process by which the innate immune system eliminates intracellular bacteria. In this study, we showed that stimulation of toll-like receptor 7 (TLR7) with imiquimod (IMQ) triggered autophagic cell death in macrophages by enhancing the generation of reactive oxygen species (ROS) via the p38- or MEK/ERK1/2-mediated signaling pathway in the early phase. IMQ significantly increased mitochondrial ROS and targeted autophagosomes to the mitochondria. Stimulation of TLR7 with IMQ enhanced the expression of BNIP3, which was localized to mitochondria and interacted with beclin-1, leading to mitophagy. In addition, IMQ substantially induced NO production through the GSK-3β-mediated signaling pathway, which led to autophagy in the late stage. We further examined whether the induction of autophagy by IMQ effectively eliminated intracellular microbes. Macrophages were infected with a virulent Mycobacterium tuberculosis (Mtb) strain, H37Rv, and then treated with IMQ. IMQ suppressed intracellular Mtb growth by inducing autophagy in a dose-dependent manner and increased NO production. Inhibition of autophagy using 3-methyladenine (3-MA) prevented autophagosome formation and control of intracellular Mtb growth in macrophages. These findings revealed a novel mechanism by which IMQ induces selective autophagy to promote intracellular killing machinery against Mtb infection in macrophages.
Highlights
Autophagy is conserved in virtually all eukaryotic cells and is a fundamental cellular homeostatic process that mediates the degradation of long-lived proteins, cellular organelles, and invading pathogens, including Mycobacterium tuberculosis (Mtb), Shigella flexneri, and Salmonella typhimurium (King, 2012; Kimmey and Stallings, 2016; Yin et al, 2016)
We verified the expression of toll-like receptor 7 (TLR7) in mouse bone marrow-derived macrophages (BMDMs), splenocytes, Raw264.7 cells and THP-1 cells, a human monocytic cell line, by RT-PCR (Supplementary Figure S1A)
We further investigated whether IMQ induced autophagic cell death in macrophages
Summary
Autophagy is conserved in virtually all eukaryotic cells and is a fundamental cellular homeostatic process that mediates the degradation of long-lived proteins, cellular organelles, and invading pathogens, including Mycobacterium tuberculosis (Mtb), Shigella flexneri, and Salmonella typhimurium (King, 2012; Kimmey and Stallings, 2016; Yin et al, 2016). The selective autophagy of mitochondria, termed mitophagy, is a quality control mechanism in which damaged or unwanted mitochondria are eliminated by autophagosomes (Kamber et al, 2015; Zaffagnini and Martens, 2016). In addition to its role in the control of damaged mitochondria, mitophagy has been associated with diverse human diseases, such as Parkinson’s disease, cancer and viral infection, in several studies (Khan et al, 2015; Gao et al, 2017; Kulikov et al, 2017). Receptor interacting protein kinase 2 (RIPK2) was shown to regulate mitophagy and the accumulation of damaged mitochondria due to downregulation of phosphorylated ULK1, which is a critical autophagy protein, during influenza A virus (IAV) infection (Lupfer et al, 2013). Mitophagy is associated with resistance to viral infection, the putative roles of mitophagy during bacterial infection are still unclear
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