TLR4-mediated autophagy in macrophages is a p62-dependent type of selective autophagy of aggresome-like induced structures (ALIS)

Autophagy plays an evolutionarily conserved role in host defense against pathogens. Autophagic protection

mechanisms against microbes range from regulating immune signaling responses to directly targeting the

pathogens for lysosomal degradation. Toll-like receptors (TLRs) that detect conserved molecular features shared by pathogens regulate several innate immune responses including autophagy. Our recent study demonstrates that autophagy reported in response to TLR4-stimulation in macrophages is selective

autophagy of aggresome-like induced structures (ALIS), and p62 (also known as SQSTM1) plays an essential role in this process. Treatment of macrophages with either Escherichia coli or lipopolysaccharide (LPS) results in the activation of nuclear factor erythroid 2-related factor 2 (Nrf2), leading to an increase in the levels of p62 mRNA and protein, assembly of ALIS and their autophagic degradation. This study revealed a signaling

role for p62, distinct from its known function as a bacterial-targeting factor, which might be critical for cellular stress response during infection.     

Induction of autophagy via innate bacterial recognition

Macroautophagy (referred to hereafter as autophagy) functions not only in self-digestion, but also in the killing and degradation of infectious pathogens in in vitro-cultured cells. Based on genetic manipulations of both the host, Drosophila and pathogen, Listeria monocytogenes, we recently reported that L. monocytogenes-induced autophagy is dependent on the recognition of the pathogen by the Drosophila pattern recognition protein, PGRP-LE. Autophagy and PGRP-LE are crucial for inhibition of the intracellular growth of bacteria in hemocytes, the target cells of L. monocytogenes infection in vivo. The importance of autophagy in the resistance of Drosophila against L. monocytogenes is further indicated in in vivo survival experiments. The signaling pathway(s) that induces autophagy by PGRP-LE is independent of the known immune signaling pathways, suggesting that another unidentified pathway(s) is involved. The results of the present study demonstrate that the induction of autophagy, as an innate immune response targeting intracellular pathogens, is activated by intracellular sensors through unidentified pathways.     

The deubiquitinating enzyme USP36 controls selective autophagy activation by ubiquitinated proteins

Initially described as a nonspecific degradation process induced upon starvation, autophagy is now known also to be involved in the degradation of specific ubiquitinated substrates such as mitochondria, bacteria and aggregated proteins, ensuring crucial functions in cell physiology and immunity. We report here that the deubiquitinating enzyme USP36 controls selective autophagy activation in Drosophila and in human cells. We show that dUsp36 loss of function autonomously inhibits cell growth while activating autophagy. Despite the phenotypic similarity, dUSP36 is not part of the TOR signaling pathway. Autophagy induced by dUsp36 loss of function depends on p62/SQSTM1, an adaptor for delivering cargo marked by polyubiquitin to autophagosomes. Consistent with p62 requirement, dUsp36 mutant cells display nuclear aggregates of ubiquitinated proteins, including Histone H2B, and cytoplasmic ubiquitinated proteins; the latter are eliminated by autophagy. Importantly, USP36 function in p62-dependent selective autophagy is conserved in human cells. Our work identifies a novel, crucial role for a deubiquitinating enzyme in selective autophagy.     

The deubiquitinating enzyme USP36 controls selective autophagy activation by ubiquitinated proteins

Initially described as a nonspecific degradation process induced upon starvation, autophagy is now known also to be involved in the degradation of specific ubiquitinated substrates such as mitochondria, bacteria and aggregated proteins, ensuring crucial functions in cell physiology and immunity. We report here that the deubiquitinating enzyme USP36 controls selective autophagy activation in Drosophila and in human cells. We show that dUsp36 loss of function autonomously inhibits cell growth while activating autophagy. Despite the phenotypic similarity, dUSP36 is not part of the TOR signaling pathway. Autophagy induced by dUsp36 loss of function depends on p62/SQSTM1, an adaptor for delivering cargo marked by polyubiquitin to autophagosomes. Consistent with p62 requirement, dUsp36 mutant cells display nuclear aggregates of ubiquitinated proteins, including Histone H2B, and cytoplasmic ubiquitinated proteins; the latter are eliminated by autophagy. Importantly, USP36 function in p62-dependent selective autophagy is conserved in human cells. Our work identifies a novel, crucial role for a deubiquitinating enzyme in selective autophagy.     

Innate immunity to mycobacteria: vitamin D and autophagy

Autophagy is an ancient mechanism of protein degradation and a novel antimicrobial strategy. With respect to host defences against mycobacteria, autophagy plays a crucial role in antimycobacterial resistance, and contributes to immune surveillance of intracellular pathogens and vaccine efficacy. Vitamin D3 contributes to host immune responses against Mycobacterium tuberculosis through LL‐37/hCAP‐18, which is the only cathelicidin identified to date in humans. In this review, we discuss recent advances in our understanding of host immune strategies against mycobacteria, including vitamin D‐mediated innate immunity and autophagy activation. This review also addresses our current understanding regarding the autophagy connection to principal innate machinery, such as ubiquitin‐ or inflammasome‐involved pathways. Integrated dialog between autophagy and innate immunity may contribute to adequate host immune defences against mycobacterial infection.     

Eating for good health: linking autophagy and phagocytosis in host defense

Autophagy is a conserved pathway that sequesters cytoplasmic material and delivers it to lysosomes for degradation. Digestion of portions of the cell interior plays a key role in the recycling of nutrients, remodeling, and disposal of superfluous organelles. Along with its metabolic function, autophagy is an important mechanism for innate immunity against invading bacteria and other pathogens. Multicellular organisms seem to have exploited autophagy to eliminate intracellular pathogens that would otherwise grow in the cytoplasm. Surprisingly, autophagy is involved in the response to extracellular pathogens as well, following their engulfment by conventional phagocytosis. Possible links between these two forms of cellular "eating" represent a new dimension in host defense.     

Selective autophagy mediated by autophagic adapter proteins

Mounting evidence suggests that autophagy is a more selective process than originally anticipated. The discovery and characterization of autophagic adapters, like p62 and NBR1, has provided mechanistic insight into this process. p62 and NBR1 are both selectively degraded by autophagy and able to act as cargo receptors for degradation of ubiquitinated substrates. A direct interaction between these autophagic adapters and the autophagosomal marker protein LC3, mediated by a so-called LIR (LC3-interacting region) motif, their inherent ability to polymerize or aggregate as well as their ability to specifically recognize substrates are required for efficient selective autophagy. These three required features of autophagic cargo receptors are evolutionarily conserved and also employed in the yeast cytoplasm-to-vacuole targeting (Cvt) pathway and in the degradation of P granules in C. elegans. Here, we review the mechanistic basis of selective autophagy in mammalian cells discussing the degradation of misfolded proteins, p62 bodies, aggresomes, mitochondria and invading bacteria. The emerging picture of selective autophagy affecting the regulation of cell signaling with consequences for oxidative stress responses, tumorigenesis and innate immunity is also addressed.     

Role and regulation of starvation-induced autophagy in the Drosophila fat body

In response to starvation, eukaryotic cells recover nutrients through autophagy, a lysosomal-mediated process of cytoplasmic degradation. Autophagy is known to be inhibited by TOR signaling, but the mechanisms of autophagy regulation and its role in TOR-mediated cell growth are unclear. Here, we show that signaling through TOR and its upstream regulators PI3K and Rheb is necessary and sufficient to suppress starvation-induced autophagy in the Drosophila fat body. In contrast, TOR's downstream effector S6K promotes rather than suppresses autophagy, suggesting S6K downregulation may limit autophagy during extended starvation. Despite the catabolic potential of autophagy, disruption of conserved components of the autophagic machinery, including ATG1 and ATG5, does not restore growth to TOR mutant cells. Instead, inhibition of autophagy enhances TOR mutant phenotypes, including reduced cell size, growth rate, and survival. Thus, in cells lacking TOR, autophagy plays a protective role that is dominant over its potential role as a growth suppressor.     

Selective autophagy mediated by autophagic adapter proteins

Mounting evidence suggests that autophagy is a more selective process than originally anticipated. The discovery and characterization of autophagic adapters, like p62 and NBR1, has provided mechanistic insight into this process. p62 and NBR1 are both selectively degraded by autophagy and able to act as cargo receptors for degradation of ubiquitinated subtstrates. A direct interaction between these autophagic adapters and the autophagosomal marker protein LC3, mediated by a so-called LIR (LC3-interacting region) motif, their inherent ability to polymerize or aggregate as well as their ability to specifically recognize substrates are required for efficient selective autophagy. These three required features of autophagic cargo receptors are evolutionarily conserved and also employed in the yeast cytoplasm-to-vacuole targeting (Cvt) pathway and in the degradation of P granules in C. elegans. Here, we review the mechanistic basis of selective autophagy in mammalian cells discussing the degradation of misfolded proteins, p62 bodies, aggresomes, mitochondria and invading bacteria. The emerging picture of selective autophagy affecting the regulation of cell signaling with consequences for oxidative stress responses, tumorigenesis and innate immunity is also addressed.     

The perplexing role of autophagy in plant innate immune responses

Autophagy is a major intracellular process for the degradation of cytosolic macromolecules and organelles in the lysosomes or vacuoles for the purposes of regulating cellular homeostasis and protein and organelle quality control. In complex metazoan organisms, autophagy is highly engaged during the immune responses through interfaces either directly with intracellular pathogens or indirectly with immune signalling molecules. Studies over the last decade or so have also revealed a number of important ways in which autophagy shapes plant innate immune responses. First, autophagy promotes defence‐associated hypersensitive cell death induced by avirulent or related pathogens, but restricts unnecessary or disease‐associated spread of cell death. This elaborate regulation of plant host cell death by autophagy is critical during plant immune responses to the types of plant pathogens that induce cell death, which include avirulent biotrophic pathogens and necrotrophic pathogens. Second, autophagy modulates defence responses regulated by salicylic acid and jasmonic acid, thereby influencing plant basal resistance to both biotrophic and necrotrophic pathogens. Third, there is an emerging role of autophagy in virus‐induced RNA silencing, either as an antiviral collaborator for targeted degradation of viral RNA silencing suppressors or an accomplice of viral RNA silencing suppressors for targeted degradation of key components of plant cellular RNA silencing machinery. In this review, we summarize this important progress and discuss the potential significance of the perplexing role of autophagy in plant innate immunity.     

Attenuation of cGAS‐STING signaling is mediated by a p62/SQSTM1‐dependent autophagy pathway activated by TBK1

Negative regulation of immune pathways is essential to achieve resolution of immune responses and to avoid excess inflammation. DNA stimulates type I IFN expression through the DNA sensor cGAS, the second messenger cGAMP, and the adaptor molecule STING. Here, we report that STING degradation following activation of the pathway occurs through autophagy and is mediated by p62/SQSTM1, which is phosphorylated by TBK1 to direct ubiquitinated STING to autophagosomes. Degradation of STING was impaired in p62‐deficient cells, which responded with elevated IFN production to foreign DNA and DNA pathogens. In the absence of p62, STING failed to traffic to autophagy‐associated vesicles. Thus, DNA sensing induces the cGAS‐STING pathway to activate TBK1, which phosphorylates IRF3 to induce IFN expression, but also phosphorylates p62 to stimulate STING degradation and attenuation of the response.     

Cigarette smoke-induced autophagy is regulated by SIRT1-PARP-1-dependent mechanism: Implication in pathogenesis of COPD

Autophagy is a fundamental cellular process that eliminates long-lived proteins and damaged organelles through lysosomal degradation pathway. Cigarette smoke (CS)-mediated oxidative stress induces cytotoxic responses in lung cells. However, the role of autophagy and its mechanism in CS-mediated cytotoxic responses is not known. We hypothesized that NAD⁺-dependent deacetylase, sirtuin 1 (SIRT1) plays an important role in regulating autophagy in response to CS. CS exposure resulted in induction of autophagy in lung epithelial cells, fibroblasts and macrophages. Pretreatment of cells with SIRT1 activator resveratrol attenuated CS-induced autophagy whereas SIRT1 inhibitor, sirtinol, augmented CS-induced autophagy. Elevated levels of autophagy were induced by CS in the lungs of SIRT1 deficient mice. Inhibition of poly(ADP-ribose)-polymerase-1 (PARP-1) attenuated CS-induced autophagy via SIRT1 activation. These data suggest that the SIRT1-PARP-1 axis plays a critical role in the regulation of CS-induced autophagy and have important implications in understanding the mechanisms of CS-induced cell death and senescence.     

A role for diacylglycerol in antibacterial autophagy

Antibacterial autophagy is understood to be a key cellular immune response to invading microbes. However, the mechanism(s) by which bacteria are selected as targets of autophagy remain unclear. We recently identified diacylglycerol as a novel signaling molecule that targets bacteria to the autophagy pathway, and show that it acts via protein kinase C activation. We also found that Pkc1 is required for autophagy in yeast, indicating that this kinase plays a conserved role in autophagy regulation.     

TLR2 mediates phagocytosis and autophagy through JNK signaling pathway in Staphylococcus aureus-stimulated RAW264.7 cells

Toll-like receptor 2 (TLR2) is involved in phagocytosis and autophagy to enhance host innate immune response to bacterial infection. TLR2 has been reported to participate in the recognition of Staphylococcus aureus (S. aureus). However, the role of TLR2 in phagocytosis and autophagy in S. aureus-stimulated macrophages and the underlying mechanisms as yet remain unclear. In the present study, stimulation of mouse macrophage cell line RAW264.7 with S. aureus activated multiple signaling pathways including mitogen-activated protein kinases (MAPKs), myeloid differentiation factor 88 (MyD88), phosphatidylinositide 3-kinase (PI3K) and Rac1 and triggered autophagy process. Knockdown of TLR2 by siRNA significantly reduced phagocytosis and autophagy of macrophages upon S. aureus infection. Interestingly, TLR2 siRNA markedly attenuated S. aureus-induced phosphorylation of c-Jun N-terminal kinase (JNK) but not p38 or extracellular regulated protein kinase (ERK) in macrophages. Similarly, SP600125, a JNK inhibitor, also down-regulated phagocytosis and autophagy in S. aureus-stimulated macrophages. Furthermore, TLR2 siRNA and SP600125 simultaneous treatment showed similar phagocytosis and autophagy compared to that in TLR2 siRNA treatment alone. Collectively, our results indicate that TLR2 may be critical for phagocytosis and autophagy through JNK signaling pathway, and provide an underlying mechanistic link between innate immune receptor and induction of phagocytosis and autophagy in S. aureus-stimulated macrophages.     

Toll-like receptors control autophagy

Autophagy is a newly recognized innate defense mechanism, acting as a cell-autonomous system for elimination of intracellular pathogens. The signals and signalling pathways inducing autophagy in response to pathogen invasion are presently not known. Here we show that autophagy is controlled by recognizing conserved pathogen-associated molecular patterns (PAMPs). We screened a PAMP library for effects on autophagy in RAW 264.7 macrophages and found that several prototype Toll-like receptor (TLR) ligands induced autophagy. Single-stranded RNA and TLR7 generated the most potent effects. Induction of autophagy via TLR7 depended on MyD88 expression. Stimulation of autophagy with TLR7 ligands was functional in eliminating intracellular microbes, even when the target pathogen was normally not associated with TLR7 signalling. These findings link two innate immunity defense systems, TLR signalling and autophagy, provide a potential molecular mechanism for induction of autophagy in response to pathogen invasion, and show that the newly recognized ability of TLR ligands to stimulate autophagy can be used to treat intracellular pathogens.