- Email: [email protected]
Regulation of inflammasomes by autophagy
Accepted Manuscript Regulation of the Inflammasomes by Autophagy Tatsuya Saitoh, PhD, Shizuo Akira, MD, PhD PII:
S0091-6749(16)30358-X
DOI:
10.1016/j.jaci.2016.05.009
Reference:
YMAI 12135
To appear in:
Journal of Allergy and Clinical Immunology
Received Date: 3 May 2016 Revised Date:
17 May 2016
Accepted Date: 18 May 2016
Please cite this article as: Saitoh T, Akira S, Regulation of the Inflammasomes by Autophagy, Journal of Allergy and Clinical Immunology (2016), doi: 10.1016/j.jaci.2016.05.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Regulation of the Inflammasomes by Autophagy
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Author Name
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Tatsuya Saitoh, PhDa* and Shizuo Akira, MD, PhDb,c
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Author Affiliation a
Department of Inflammation Biology, Institute for Advanced Enzyme Research, Tokushima
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University, Tokushima 770-8503, Japan
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b
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Laboratory of Host Defense, World Premier International Research Center Immunology Frontier
Research Center, Osaka University, Osaka 565-0871, Japan
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c
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565-0871, Japan
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Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka
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Tatsuya Saitoh, PhD, Department of Inflammation Biology, Institute for Advanced Enzyme Research,
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Tokushima University, Tokushima 770-8503, Japan. Email: [email protected]
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Competing Interests
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The authors have declared that no competing interests exist.
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Grant Support
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This work was partly supported by Japan Society for the Promotion of Science KAKENHI (Grant
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Numbers 15H01380, 26111514, 26713005 to T.S.); Japan Agency for Medical Research and
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Development, Core Research for Evolutional Science and Technology (to T.S.); Research Grant of
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the Japan Foundation for Pediatric Research (to T.S.); Research Grant of the LOTTE Foundation (to
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T.S.); Research Grant of the Takeda Science Foundation (to T.S.); Research Grant of the Mochida
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Memorial Foundation for Medical and Pharmaceutical Research (to T.S.); Research Grant of the
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Naito Foundation (to T.S.); and Research Grant of the Tokyo Biochemical Research Foundation (to
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T.S.).
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37 Abstract
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Inflammasomes detect pathogen-associated molecular patterns to induce inflammatory innate
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immune responses and play a key role in host defense against infectious agents. However,
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inflammasomes are often wrongly activated by metabolites, amyloids, and environmental irritants.
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This induces massive inflammation, causing severe tissue damage and results in the development of
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inflammatory diseases. Hence, cellular machineries regulating both “activation” and “inactivation”
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of inflammasomes are definitely important. Recent studies have shown that autophagy, an
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intracellular degradation system associated with maintenance of cellular homeostasis, plays a key
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role in inflammasome inactivation. Notably, autophagy-deficiency caused by gene mutation disrupts
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organelle elimination and thus induces aberrant activation of the inflammasomes, leading to severe
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tissue damage. Here, we review recent findings regarding the involvement of autophagy in the
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regulation of inflammasome activation and development of inflammatory disorders.
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50 51 52 Key words
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autophagy; host defense; inflammasome; inflammatory disorders; innate immunity; macrophages;
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organelle; pattern-recognition receptors; reactive oxygen species; signal transduction
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Abbreviations
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AIM2: absent in melanoma 2; ATG proteins: autophagy-related proteins; ATP: Adenosine
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triphosphate; ASC: apoptosis-associated speck-like protein containing caspase recruitment domain;
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DSS: dextran sulfate sodium; ER: endoplasmic reticulum; IFN: interferon; IL: interleukin; LPS:
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lipopolysaccharide; MSU: monosodium urate; NLR: NOD-like receptors; NLRP3: NLR family,
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pyrin domain containing 3; PRR: pattern-recognition receptor; ROS: reactive oxygen species; TLR:
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Toll-like receptor
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73 Introduction
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Autophagy is an intracellular degradation system that delivers cytoplasmic constituents into
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lysosome.1,2. Autophagy activity is maintained at relatively low levels under steady-state conditions,
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but is potently induced by various cellular stressors such as organelle damage and pathogen infection.
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The autophagosome, a double-membraned organelle, plays an important role in autophagic
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degradation. Autophagy induction is accompanied by the emergence of the isolation membrane, also
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called the phagophore. Endoplasmic reticulum and mitochondria, especially mitochondria-associated
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endoplasmic reticulum membrane, provide a membrane source for the isolation membrane. The
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autophagosome, formed by elongation and closure of the isolation membrane engulfs a portion of
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the cytoplasm and subsequently, fuses with the lysosome to form the autolysosome. This leads to
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degradation of the autolysosome contents as well as the inner membrane by lysosomal degradation
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enzymes (Figure 1).
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Accumulating evidence shows that autophagy is involved in various biological
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processes.3-5 Steady-state autophagy promotes the turnover of organelles such as mitochondria and
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contributes to the maintenance of cellular homeostasis. Autophagy is induced under nutrient-starved
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conditions and contributes to reuse of cytoplasmic constituents such as amino acids and lipids.
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Autophagy is also induced by organelle stress and pathogen infection, contributing to the elimination
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of damaged organelles and pathogens. Additionally, autophagy regulates cell conditions, death,
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differentiation, and tumorigenesis, and is closely associated with high-order cell functions such as
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immune response and host defense.
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The innate immune system is activated after infection by pathogens and induces
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inflammation to protect the host.6 Pattern-recognition receptors (PRRs) are key players of the innate
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immune system, sensing microbial components such as lipopolysaccharide and flagellin, and play a
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critical role in induction of inflammatory response. After sensing microbial components, PRRs
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induce signal transduction pathways that cause activation of transcription factors such as nuclear
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factor-κB and interferon regulatory factors. These transcription factors induce expression of
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inflammatory mediators such as cytokines, chemokines, and type I interferons (IFNs). PRRs also
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induce activation of inflammatory proteases leading to production of processed mature cytokines.
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While inflammation is essential for host defense, wrongly induced inflammation often causes the
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development of inflammatory diseases such as septic shock, autoimmune diseases, and metabolic
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diseases.7 Therefore, rigorous control of the innate immune system is required to prevent insufficient
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or excessive inflammatory responses. A growing body of evidence has shown that intracellular
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degradation systems play an important role in this regulation, where autophagy has been recognized
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as critically involved in innate immune responses mediated by toll-like receptors (TLRs), retinoic
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acid-inducible gene-I-like receptor, cyclic guanosine monophosphate-adenosine monophosphate
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synthase, and inflammasomes.4,8-18 In this review, we discuss recent advances in the understanding
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of autophagy and inflammatory innate immune response, especially inflammasome activation.
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Identification and function of autophagy-related proteins
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Autophagic structures were first reported through electron microscopy studies in the 1950s and
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contributors to autophagosome formation were identified via yeast genetic screening in the
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1990s.19-22 Essential components of autophagosome formation are called as autophagy-related
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proteins (ATG proteins). At present, 38 ATG proteins have been identified in yeast. Core ATG
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proteins are phylogenetically highly conserved. Mammalian counterparts such as ULK1
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(mammalian ATG1), ATG3, ATG4s, ATG5, BECLIN1 (mammalian ATG6), ATG7, LC3A/B
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(mammalian ATG8), ATG9A, ATG10, ATG12, ATG13, ATG14, ATG16L1, FIP200 (mammalian
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ATG17), and WIPIs (mammalian ATG18) have been identified. Hence, autophagy is an
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evolutionarily conserved machinery for maintenance of cellular homeostasis. Core ATG proteins are
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classified into several functional units: the FIP200 complex; the class III PI3K complex; the
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ATG7-mediated, ubiquitin-like conjugation system; etc. (Figure 1). The coordinated action of
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functional ATG protein units induces the membrane trafficking events responsible for isolation
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membrane formation and subsequent autophagosome formation. Mice lacking ATG3, ATG5, ATG7,
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or ATG16L1 have been observed to die shortly after birth because of poor nutrition and energy
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depletion; therefore, autophagy-dependent maintenance of cellular homeostasis is required for
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survival. On the other hand, mice lacking BECLIN1 and FIP200 are embryonic lethal. Thus, ATG
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proteins have additional functions beyond simply inducing autophagosome formation.
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Selective clearance of damaged organelles and ubiquitinated proteins by autophagy
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Autophagy has been considered as a non-specific bulk degradation process. However, at present, it is
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clear that autophagy selectively eliminates damaged organelles, pathogen-containing vacuoles, and
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insoluble protein aggregates.18,23 The initial event that induces selective autophagy is ubiquitination
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of the substrate. Ubiquitination is a hallmark of unfavorable material within the cell and the
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autophagy system seeks to eliminate such material. The autophagosome engulfs damaged organelles
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and pathogen-containing vacuole that contain ubiquitinated membranes and ubiquitinated protein
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aggregates, for further degradation within the lysosome. Recent studies have identified p62, NDP52,
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and optineurin as the adaptor molecules that bind to ubiquitinated proteins and recruit ATG proteins
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to induce selective degradation of substrates by autophagy (Figure 2). As β-galactose chains
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normally exist in the lumen of endosomal and phagosomal compartments, exposure of β-galactose
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chains to cytosolic galectins is other hallmark of damaged endosomes and phagosomes. Adaptor
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protein, NDP52 binds to not only ubiquitin chains but also galectin-8, a cytosolic galectin, to recruit
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autophagy machinery toward the damaged endosome and phagosome. Thus, selective autophagy
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maintains cellular homeostasis by eliminating unfavorable material within the cell.
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The inflammasome, an innate immune structure, is mainly activated in myeloid cells such as
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macrophages and induces inflammation to protect the host from microbial infection.24,25 The
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inflammasome is a cytosolic protein complex and consists of PRRs such as NOD-like receptors
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(NLRs) and absent in melanoma 2 (AIM2), together with the adaptor protein, apoptosis-associated
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speck-like protein containing caspase recruitment domain (ASC), and the protease caspase-1.
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Activation of the inflammasome induces processing of pro-interleukin (IL)-1β and pro-IL-18 by
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caspase-1, which in turn induces production of these cytokines. Inflammasome-dependent cytokine
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production plays an important role in host defense against microbes. However, inflammasome is
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often wrongly activated by host-derived stimulatory factors and environmental irritants, and results
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in massive inflammation. Disruption of negative regulatory mechanisms and exposure to excess
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microbial components also cause massive inflammation, which in turn results in severe tissue
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damage, and subsequently the development of infectious and inflammatory disorders.
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ATG16L1 in lipopolysaccharide-induced inflammation
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Genome-wide association studies have revealed that single nucleotide polymorphisms of the gene
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encoding ATG16L1, an autophagy-related protein, is associated with susceptibility to Crohn's
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disease, a chronic inflammatory disease of the intestine.26-27 Mouse colitis models have confirmed
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that ATG16L1 is critical in maintenance of intestinal homeostasis and indicate that ATG16L1 is a
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genetic factor responsible for development of colitis. Hematopoietic cell-specific ATG16L1-deficient
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mice and mice expressing Crohn’s disease-associated ATG16L1 variant (T300A) are highly sensitive
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to experimental colitis induced by dextran sulfate sodium (DSS) and pathogenic bacteria.11,28,29
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Macrophages lacking ATG16L1 and expressing Crohn’s disease-associated ATG16L1 variant
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produce large quantities of IL-1β and IL-18 in response to lipopolysaccharide (LPS) (Figure 3).
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Importantly, peripheral blood mononuclear cells from Crohn's disease patients expressing Crohn’s
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disease-associated ATG16L1 variant also produce large quantities of IL-1β.28,29 Upon stimulation,
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apoptotic proteases caspase-3 and -7 induce proteolytic cleavage of the Crohn’s disease-associated
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ATG16L1 variant, resulting in destabilization of ATG16L1 (Figure 4).28,29 Thus, apoptotic caspases
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enhance production of IL-1β in macrophages expressing Crohn’s disease-associated ATG16L1
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variant. Macrophages lacking ATG7 or treated with VPS34 inhibitor, 3-methyladenine produce large
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quantities of IL-1β in response to LPS, indicating that multiple autophagy-related proteins are
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involved in the response.11 Autophagy is therefore, involved in LPS-induced IL-1β production,
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though this is not a unique function of ATG16L1. On the other hand, ATG16L1 in intestinal
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epithelial cells is also important in the control of intestinal inflammation. Hypomorphic mutation of
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ATG16L1 gene in mice causes dysfunction of Paneth cells, a specialized epithelial cell, as well as
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elevated production of tumor necrosis factor and IFN-γ, resulting in enhanced susceptibility to
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DSS-induced experimental colitis.30,31 Dysfunction of Paneth cells is also found in intestinal
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epithelial cell-specific, ATG5- and ATG7-deficient mice and is caused by defective host defense
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against norovirus. Thus, autophagy is involved in the prevention of aberrant inflammation and in
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maintenance of intestinal homeostasis.
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NLR family, pyrin domain containing 3 (NLRP3) is a well-characterized PRR belonging to the
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NOD-like receptor family.24,25 NLRP3 forms the inflammasome complex with adaptor protein, ASC
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and
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NLRP3-inflammasome is activated after exposure to various organelle stressors in myeloid cells
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such as macrophages. Damage of phagosome-lysosome, mitochondria and endoplasmic reticulum
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(ER) potently activates NLRP3-inflammasome and induces production of IL-1β and IL-18.32-36
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Reactive oxygen species (ROS) and mitochondrial DNA released from damaged mitochondria play a
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key role in activation of the NLRP3-inflammasome.32-37 Phagosomal rupture and ER stress cause
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mitochondrial damage. Adenosine triphosphate (ATP), a danger signal produced from dying cells,
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causes mitochondrial damage to activate NLRP3-inflammasome, resulting in elimination of
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unfavorable, accumulated, dead cells via inflammation. NLRP3-inflammasome detects phagosomal
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rupture caused by bacterial lysins such as streptolysin and hemolysin, and induces inflammation to
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protect the host from bacterial infection. NLRP3-inflammasome is wrongly activated by stimulatory
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metabolites such as monosodium urate (MSU) crystals, cholesterol crystals, and palmitic acid, which
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in turn induces severe tissue damage, resulting in development of metabolic diseases.
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NLRP3-inflammasome is also wrongly activated by environmental irritants such as asbestos and
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silica particles, leading to severe lung tissue damage.
induce maturation
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protease caspase-1
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Autophagy promotes turnover of organelles, thus preventing the accumulation of highly
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fragile, aging organelles as well as elimination of damaged organelles.1,2,18,23 Thus, autophagy
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suppresses organelle stress-induced activation of the NLRP3-inflammasome. As expected, in
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autophagy-deficient macrophages, damaged mitochondria accumulate and produce excessive
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amounts of ROS after stimulation by inducers such as MSU crystals, cholesterol crystals and
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palmitic acid, resulting in the enhanced activation of the NLRP3-inflammasome (Figure 5).11,34,37-40
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Accumulated
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damaged
mitochondria
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mitochondrial DNA, a inducer of the NLRP3-inflammasome.
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macrophages
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Adaptor protein p62 recognizes 6
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damaged mitochondria, where membrane proteins are ubiquitinated by PARKIN, and induces
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selective autophagy.41 Amino acid sensor general control non-derepressible-2-dependent autophagy
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in intestinal antigen-presenting cells suppresses inflammasome activation and is critical for
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prevention of intestinal inflammation.42 During influenza A virus infection, nucleotide-binding
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oligomerization domain containing protein 2 detects viral RNA and promotes autophagic elimination
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of damaged mitochondria by inducing ULK1 phosphorylation and limits excessive activation of the
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NLRP3-inflammasome
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NLRP3-inflammasome-dependent inflammation and leads to exacerbation of various inflammatory
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disorders such as sepsis, colitis, pneumonia, atherosclerosis, and diabetes.11,37-43 On the other hand,
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inducers
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NLRP3-inflammasome and alleviate inflammation-induced tissue damage.44-46
autophagy
suppress
6).43
Loss
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of
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IL-1β
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IL-18
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Autophagy in non-canonical inflammasome activation
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Recent studies have identified that cytosolic LPS stimulates the non-canonical inflammasome to
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induce pyroptosis and production of IL-1α, IL-1β, and IL-18 in myeloid cells such as
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macrophages.25,47 Non-canonical inflammasome in mouse consists of caspase-11, whereas, in human,
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it consists of caspase-4 and -5. Although TLR4 is required for inducible expression of caspase-11 as
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well as IL-1α and IL-1β, it is not involved in recognition of cytosolic LPS. During infection by
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gram-negative bacteria, guanylate-binding proteins are transcriptionally induced by TLRs and
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promote membrane rupture of the bacteria-containing phagosome.48 Membrane rupture of
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bacteria-containing phagosome releases bacterial LPS into the cytosol, thereby facilitating detection
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of LPS by the non-canonical inflammasome. Autophagy deficiency causes accumulation of the
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damaged bacteria-containing phagosome and greatly enhances activation of the non-canonical
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inflammasome. Sensor proteins such as NDP52 recognize ubiquitin chains and galectin-8 in
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damaged bacteria-containing phagosome, and induce selective autophagy. Thus, autophagy
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eliminates bacteria-containing phagosome and prevents excessive induction of non-canonical
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inflammasome-mediated inflammation (Figure 7).
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Autophagic degradation of ASC and IL-1β β
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Autophagy suppresses activation of the AIM2- and NLRP3-inflammasomes. Inducers of these
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inflammasomes cause lysine 63-linked ubiquitination of ASC, an adaptor molecule of
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inflammasomes.49 The ubiquitin sensor p62 binds to ubiquitinated ASC and induces its degradation
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by inducing selective autophagy, resulting in suppression of inflammasome activation (Figure 8).
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Furthermore, the autophagosome engulfs IL-1β and delivers it into lysosomal compartments for
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degradation (Figure 8).50 Hence, autophagy disrupts multiple steps of inflammasome activation to
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prevent excessive inflammation. 7
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253 Autophagy in IL-1β β secretion
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Autophagy suppresses activation of the inflammasome under normal and nutrient-rich conditions.
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However, a recent study has shown that autophagy is often involved in the unconventional secretion
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of IL-1β under nutrient-starved conditions (Figure 9)51. Macrophages lacking ATG5 produce scarce
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amounts of IL-1β in response to nigericin, an inducer of the NLRP3-inflammasome under
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nutrient-starved conditions. Golgi reassembly stacking protein and RAB8A, membrane traffic
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regulators, are also involved in nigericin-induced secretion of IL-1β. On the other hand, other studies
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have shown that autophagy limits production of IL-1β under nutrient-starved conditions.49 At present,
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differences in conditions that can account for this apparent discrepancy are not clarified. Hence,
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further study is needed to define the role of autophagy in IL-1β production under nutrient-starved
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conditions.
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Recent studies have revealed the involvement of autophagy in inflammatory innate immune response.
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In myeloid cells such as macrophages, autophagy suppresses inflammasome activation by promoting
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elimination of hazardous material such as damaged organelles.11,37-43,48 Autophagy deficiency in
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myeloid cells causes aberrant activation of inflammasome, leading to development of inflammatory
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disorders (Figure 10). Therefore, autophagy would be a promising therapeutic target for treatment of
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inflammasome-related inflammatory disorders. Therapeutic agents capable of inducing autophagy
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should be developed to alleviate inflammasome-dependent inflammation.
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Since the discovery of autophagy-related proteins, the role of autophagy in immune
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response has been extensively investigated.4,18 Autophagy is involved not only in PRR-mediated
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inflammation, but also in various immune responses such as pathogen elimination, antigen
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presentation, and immune cell development. However, the mechanism/s underlying autophagic
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control of the immune response are yet unclear. Future investigation will clarify the fundamental role
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of autophagy in immune response and provide a molecular basis for innovative drug development.
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24. Latz E, Xiao TS, Stutz A. Activation and regulation of the inflammasomes. Nat Rev Immunol. 2013;13:397-411. 25. Man SM, Kanneganti TD. Converging roles of caspases in inflammasome activation, cell death and innate immunity. Nat Rev Immunol. 2016;16:7-21. 26. Hampe J, Franke A, Rosenstiel P, Till A, Teuber M, Huse K, et al. A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1. Nat Genet. 2007;39:207-211. 27. Rioux JD, Xavier RJ, Taylor KD, Silverberg MS, Goyette P, Huett A, et al. Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis. Nat Genet. 2007;39:596-604. 28. Lassen KG, Kuballa P, Conway KL, Patel KK, Becker CE, Peloquin JM, et al. Atg16L1 T300A variant decreases selective autophagy resulting in altered cytokine signaling and decreased antibacterial defense. Proc Natl Acad Sci U S A. 2014;111:7741-7746. 29. Murthy A, Li Y, Peng I, Reichelt M, Katakam AK, Noubade R, et al. A Crohn’s disease variant in Atg16l1 enhances its degradation by caspase 3. Nature. 2014;506:456-462. 30. Cadwell K, Liu JY, Brown SL, Miyoshi H, Loh J, Lennerz JK, et al. A key role for autophagy and the autophagy gene Atg16l1 in mouse and human intestinal Paneth cells. Nature. 2008;456:259-263. 31. Cadwell K, Patel KK, Maloney NS, Liu TC, Ng ACY, Storer CE, et al. Virus-plus-susceptibility gene interaction determines Crohn’s disease gene Atg16L1 phenotypes in intestine. Cell. 2010;141:1135-45. 32. Dostert C, Pétrilli V, Van Bruggen R, Steele C, Mossman BT, Tschopp J. Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica. Science. 2008;320:674-7. 33. Hornung V, Bauernfeind F, Halle A, Samstad EO, Kono H, Rock KL, et al. Silica crystals and aluminum salts activate the NALP3 inflammasome through phagosomal destabilization. Nat Immunol. 2008;9:847-56. 34. Zhou R, Yazdi AS, Menu P, Tschopp J. A role for mitochondria in NLRP3 inflammasome activation. Nature. 2011;469:221-225. 35. Misawa T, Takahama M, Kozaki T, Lee H, Zou J, Saitoh T, et al. Microtubule-driven spatial arrangement of mitochondria promotes activation of the NLRP3 inflammasome. Nat Immunol. 2013;14:454-60. 36. Bronner DN, Abuaita BH, Chen X, Fitzgerald KA, Nuñez G, He Y, et al. Endoplasmic Reticulum Stress Activates the Inflammasome via NLRP3- and Caspase-2-Driven Mitochondrial Damage. Immunity. 2015;43:451-62. 37. Nakahira K, Haspel JA, Rathinam VA, Lee SJ, Dolinay T, Lam HC, et al. Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome. Nat Immunol. 2011;12:222-230. 38. Wen H, Gris D, Lei Y, Jha S, Zhang L, Huang MT, et al. Fatty acid-induced NLRP3-ASC inflammasome activation interferes with insulin signaling. Nat Immunol. 2011;12:408-15. 39. Razani B, Feng C, Coleman T, Emanuel R, Wen H, Hwang S, et al. Autophagy links inflammasomes to atherosclerotic progression. Cell Metab. 2012;15:534-544. 40. Lim YM, Lim H, Hur KY, Quan W, Lee HY, Cheon H, et al. Systemic autophagy insufficiency compromises adaptation to metabolic stress and facilitates progression from obesity to diabetes. Nat Commun. 2014;5:4934. 41. Zhong Z, Umemura A, Sanchez-Lopez E, Liang S, Shalapour S, Wong J, et al. NF-κB Restricts
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Inflammasome Activation via Elimination of Damaged Mitochondria. Cell. 2016;164:896-910. Ravindran R, Loebbermann J, Nakaya HI, Khan N, Ma H, Gama L, et al. The amino acid sensor GCN2 controls gut inflammation by inhibiting inflammasome activation. Nature. 2016;531:523-7. Lupfer C, Thomas PG, Anand PK, Vogel P, Milasta S, Martinez J, et al. Receptor interacting protein kinase 2-mediated mitophagy regulates inflammasome activation during virus infection. Nat Immunol. 2013;14:480-488. Shaw SY, Tran K, Castoreno AB, Peloquin JM, Lassen KG, Khor B, et al. Selective modulation of autophagy, innate immunity, and adaptive immunity by small molecules. ACS Chem Biol. 2013;8:2724-2733. Guo W, Sun Y, Liu W, Wu X, Guo L, Cai P, et al. Small molecule-driven mitophagy-mediated NLRP3 inflammasome inhibition is responsible for the prevention of colitis-associated cancer. Autophagy. 2014;10:972-985. Abderrazak A, Couchie D, Mahmood DF, Elhage R, Vindis C, Laffargue M, et al. 2015. Anti-Inflammatory and Anti-Atherogenic Effects of the Inflammasome NLRP3 Inhibitor, Arglabin, in ApoE2.Ki Mice Fed a High Fat Diet. Circulation. 2015;131:1061-70. Lamkanfi M, Dixit VM. Mechanisms and functions of inflammasomes. Cell. 2014;157:1013-1022. Meunier E, Dick MS, Dreier RF, Schürmann N, Kenzelmann Broz D, Warming S, et al. Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-induced GTPases. Nature. 2014;509:366-370. Shi CS, Shenderov K, Huang NN, Kabat J, Abu-Asab M, Fitzgerald KA, et al. Activation of autophagy by inflammatory signals limits IL-1β production by targeting ubiquitinated inflammasomes for destruction. Nat Immunol. 2012;13:255-263. Harris J, Hartman M, Roche C, Zeng SG, O'Shea A, Sharp FA, et al. Autophagy controls IL-1beta secretion by targeting pro-IL-1beta for degradation. J Biol Chem. 2011;286:9587-9597. Dupont N, Jiang S, Pilli M, Ornatowski W, Bhattacharya D, Deretic V. Autophagy-based unconventional secretory pathway for extracellular delivery of IL-1β. EMBO J. 2011;30:4701-4711.
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Figure Legends
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Fig.1. Autophagy.
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The isolation membrane appears after exposure to stressors. Endoplasmic reticulum-mitochondria
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contact site is a membrane source for the isolation membrane. The isolation membrane elongates
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and engulfs cytosolic constituents to form the autophagosome, which fuses with lysosomes to form
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the autolysosome, resulting in degradation of the engulfed constituents. Autophagy promotes the
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reuse of cellular components, turnover of old organelles and elimination of unfavorable materials,
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thus playing a vital role in the maintenance of cellular homeostasis and prevention of disease.
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Autophagy-related proteins drive membrane trafficking necessary for the generation of isolation
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membrane and autophagosomes. 11
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425 Fig.2. Autophagy eliminates damaged organelles.
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Membranes of damaged phagosomes containing bacteria or crystals and damaged mitochondria are
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ubiquitinated and targeted by adaptor proteins such as p62, NDP52 and optineurin. β-galactose
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chains within the damaged phagosome are recognized by cytosolic galectin-8, which binds to
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adaptor protein NDP52. Adaptor proteins recruit autophagy-related proteins to generate
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autophagosomes in the proximity of the damaged organelle, which engulf and eliminate damaged
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organelles after fusion with lysosomes.
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Fig.3. Autophagy suppresses TLR4-dependent production of IL-1β β and IL-18.
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TLR4 detects LPS, triggers MyD88-dependent signaling pathway to activate the transcription factor,
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nuclear factor-κB, and promotes pro-IL-1β expression. (A) TLR4 hardly induces production of
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IL-1β and IL-18 in autophagy-competent normal macrophages, because autophagy suppresses
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TRIF-dependent signaling pathway leading to ROS generation. (B) TRIF-mediated ROS production
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is greatly enhanced in autophagy-deficient macrophages, which lack ATG16L1 expression. Thus,
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autophagy-deficiency enhances LPS-induced production of IL-1β and IL-18.
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Fig.4. Crohn’s disease-associated ATG16L1 is susceptible to protease degradation.
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Crohn’s disease-associated ATG16L1 variant (T300A), but not wild-type ATG16L1, is processed by
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caspase-3 and -7. When cells are exposed to severe stress, these caspases induce processing of
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ATG16L1 (T300A) and make it unstable, resulting in loss of autophagy activity. Autophagy
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deficiency causes massive inflammation leading to development of colitis.
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Fig.5. Autophagy suppresses NLRP3-inflammasome activation.
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Stimulatory particles such as MSU and cholesterol crystals cause phagosomal rupture, resulting in
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mitochondrial damage. ATP released from dying cells stimulates P2X7R and causes mitochondrial
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damage, which in turn releases ROS, causing activation of the NLRP3-inflammasome.
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Mitochondrial damage also causes release of mitochondrial DNA (mtDNA) to activate the
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NLRP3-inflmmasome. PARKIN, an E3 ubiquitin ligase, induces ubiquitination of a set of proteins
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expressed on damaged mitochondria. Autophagy selectively eliminates damaged mitochondria after
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sensing ubiquitinated proteins by adaptor protein p62. Autophagy suppresses activation of the
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NLRP3-inflammasome by eliminating damaged mitochondria, which prevents excess production of
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ROS and mtDNA.
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Fig.6. NOD2-dependent autophagy suppresses influenza virus-induced activation of the
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NLRP3-inflammasome. 12
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Influenza virus infection causes mitochondrial damage, causing ROS production, that results in
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NLRP3-inflammasome activation. Autophagy eliminates damaged mitochondria to prevent excess
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ROS production and subsequent activation of the NLRP3-inflammasome. However, influenza virus
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also induces autophagy by stimulating NOD2. NOD2 deficiency causes impaired autophagy
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induction, resulting in massive pulmonary inflammation during influenza virus infection.
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Fig.7. Autophagy suppresses non-canonical inflammasome activation.
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Gram-negative bacteria stimulate TLR4 via LPS and induce expression of guanylate-binding
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proteins, caspase-11, pro-IL-1α and pro-IL-1β in mouse macrophages. Gram-negative bacteria also
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induce phagosomal rupture, causing leakage of LPS into the cytosol, which then stimulates
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caspase-11-driven non-canonical pathway of inflammasome activation. This leads to induction of
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pyroptosis and production of IL-1α and IL-1β. Autophagy eliminates damaged phagosomes to
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prevent aberrant activation of the NLRP3-inflammasome.
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Fig.9. Autophagy mediates IL-1β β secretion under starved condition.
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Under starved conditions, autophagy-related protein ATG5 is involved in secretion of IL-1β induced
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by nigericin, an inducer of the NLRP3-inflammasome. Golgi reassembly stacking protein and
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RAB8A are also involved in IL-1β secretion.
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Fig.8. Autophagy suppresses inflammatory response by eliminating of ASC and IL-1β β.
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Ubiquitinated ASC is targeted by adaptor proteins p62 and is recruited to the autophagosome. IL-1β
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is detected in LC3-positive cytosolic compartment. Autophagosome engulfs these substrates and
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eliminates them after fusion with lysosome.
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Fig.10. Autophagy deficiency causes inflammasome-related inflammatory disorders.
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Autophagy deficiency caused by gene disruption results in accumulation of damaged organelles,
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ASC, and IL-1β after exposure to various stressors such as microbes and stimulatory metabolites.
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Thus, autophagy deficiency induces aberrant activation of the inflammasomes and subsequent
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production of IL-1β and IL-18, leading to the development of inflammatory disorders.
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Lysosome Isolation membrane
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Autophagosome Fusion
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• Ubiquitin-like conjugation system ATG7, ATG10, ATG12, ATG5, ATG16L1, ATG3, LC3A/B, ATG4s (mATG8) • PI3K complex VPS34, VPS15, BECLIN1, ATG14 (mATG6) • FIP200 complex FIP200, ATG13, ATG101, ULK1/2 (mATG17) (mATG1) • Others ATG9A, ATG2A/B, WIPIs (mATG18)
Roles Reuse of cellular components Promotion of cellular turnover Elimination of damaged organelle Elimination of invading pathogens
Immune response Host defense Cell death / differentiation Tumorigenesis Nutrient metabolism
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Autophagosome
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Ub Ub Ub Ub
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Bacteria
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LPS TLR4
MyD88
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TLR4
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WD repeat domains
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MSU crystals Cholesterol crystals
ATP Dying cells
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Influenza virus
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IL-1β
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Bacteria
Inflammatory response
Inflammatory disorders Colitis
Accumulation of damaged organelles and signal regulators
Sepsis
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S0091-6749(16)30358-X
DOI:
10.1016/j.jaci.2016.05.009
Reference:
YMAI 12135
To appear in:
Journal of Allergy and Clinical Immunology
Received Date: 3 May 2016 Revised Date:
17 May 2016
Accepted Date: 18 May 2016
Please cite this article as: Saitoh T, Akira S, Regulation of the Inflammasomes by Autophagy, Journal of Allergy and Clinical Immunology (2016), doi: 10.1016/j.jaci.2016.05.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Regulation of the Inflammasomes by Autophagy
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Author Name
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Tatsuya Saitoh, PhDa* and Shizuo Akira, MD, PhDb,c
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Author Affiliation a
Department of Inflammation Biology, Institute for Advanced Enzyme Research, Tokushima
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University, Tokushima 770-8503, Japan
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b
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Laboratory of Host Defense, World Premier International Research Center Immunology Frontier
Research Center, Osaka University, Osaka 565-0871, Japan
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c
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565-0871, Japan
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Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka
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Tatsuya Saitoh, PhD, Department of Inflammation Biology, Institute for Advanced Enzyme Research,
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Tokushima University, Tokushima 770-8503, Japan. Email: [email protected]
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Competing Interests
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The authors have declared that no competing interests exist.
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Grant Support
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This work was partly supported by Japan Society for the Promotion of Science KAKENHI (Grant
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Numbers 15H01380, 26111514, 26713005 to T.S.); Japan Agency for Medical Research and
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Development, Core Research for Evolutional Science and Technology (to T.S.); Research Grant of
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the Japan Foundation for Pediatric Research (to T.S.); Research Grant of the LOTTE Foundation (to
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T.S.); Research Grant of the Takeda Science Foundation (to T.S.); Research Grant of the Mochida
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Memorial Foundation for Medical and Pharmaceutical Research (to T.S.); Research Grant of the
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Naito Foundation (to T.S.); and Research Grant of the Tokyo Biochemical Research Foundation (to
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T.S.).
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37 Abstract
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Inflammasomes detect pathogen-associated molecular patterns to induce inflammatory innate
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immune responses and play a key role in host defense against infectious agents. However,
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inflammasomes are often wrongly activated by metabolites, amyloids, and environmental irritants.
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This induces massive inflammation, causing severe tissue damage and results in the development of
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inflammatory diseases. Hence, cellular machineries regulating both “activation” and “inactivation”
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of inflammasomes are definitely important. Recent studies have shown that autophagy, an
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intracellular degradation system associated with maintenance of cellular homeostasis, plays a key
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role in inflammasome inactivation. Notably, autophagy-deficiency caused by gene mutation disrupts
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organelle elimination and thus induces aberrant activation of the inflammasomes, leading to severe
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tissue damage. Here, we review recent findings regarding the involvement of autophagy in the
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regulation of inflammasome activation and development of inflammatory disorders.
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50 51 52 Key words
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autophagy; host defense; inflammasome; inflammatory disorders; innate immunity; macrophages;
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organelle; pattern-recognition receptors; reactive oxygen species; signal transduction
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Abbreviations
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AIM2: absent in melanoma 2; ATG proteins: autophagy-related proteins; ATP: Adenosine
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triphosphate; ASC: apoptosis-associated speck-like protein containing caspase recruitment domain;
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DSS: dextran sulfate sodium; ER: endoplasmic reticulum; IFN: interferon; IL: interleukin; LPS:
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lipopolysaccharide; MSU: monosodium urate; NLR: NOD-like receptors; NLRP3: NLR family,
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pyrin domain containing 3; PRR: pattern-recognition receptor; ROS: reactive oxygen species; TLR:
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Toll-like receptor
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73 Introduction
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Autophagy is an intracellular degradation system that delivers cytoplasmic constituents into
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lysosome.1,2. Autophagy activity is maintained at relatively low levels under steady-state conditions,
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but is potently induced by various cellular stressors such as organelle damage and pathogen infection.
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The autophagosome, a double-membraned organelle, plays an important role in autophagic
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degradation. Autophagy induction is accompanied by the emergence of the isolation membrane, also
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called the phagophore. Endoplasmic reticulum and mitochondria, especially mitochondria-associated
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endoplasmic reticulum membrane, provide a membrane source for the isolation membrane. The
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autophagosome, formed by elongation and closure of the isolation membrane engulfs a portion of
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the cytoplasm and subsequently, fuses with the lysosome to form the autolysosome. This leads to
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degradation of the autolysosome contents as well as the inner membrane by lysosomal degradation
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enzymes (Figure 1).
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Accumulating evidence shows that autophagy is involved in various biological
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processes.3-5 Steady-state autophagy promotes the turnover of organelles such as mitochondria and
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contributes to the maintenance of cellular homeostasis. Autophagy is induced under nutrient-starved
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conditions and contributes to reuse of cytoplasmic constituents such as amino acids and lipids.
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Autophagy is also induced by organelle stress and pathogen infection, contributing to the elimination
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of damaged organelles and pathogens. Additionally, autophagy regulates cell conditions, death,
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differentiation, and tumorigenesis, and is closely associated with high-order cell functions such as
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immune response and host defense.
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The innate immune system is activated after infection by pathogens and induces
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inflammation to protect the host.6 Pattern-recognition receptors (PRRs) are key players of the innate
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immune system, sensing microbial components such as lipopolysaccharide and flagellin, and play a
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critical role in induction of inflammatory response. After sensing microbial components, PRRs
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induce signal transduction pathways that cause activation of transcription factors such as nuclear
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factor-κB and interferon regulatory factors. These transcription factors induce expression of
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inflammatory mediators such as cytokines, chemokines, and type I interferons (IFNs). PRRs also
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induce activation of inflammatory proteases leading to production of processed mature cytokines.
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While inflammation is essential for host defense, wrongly induced inflammation often causes the
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development of inflammatory diseases such as septic shock, autoimmune diseases, and metabolic
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diseases.7 Therefore, rigorous control of the innate immune system is required to prevent insufficient
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or excessive inflammatory responses. A growing body of evidence has shown that intracellular
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degradation systems play an important role in this regulation, where autophagy has been recognized
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as critically involved in innate immune responses mediated by toll-like receptors (TLRs), retinoic
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acid-inducible gene-I-like receptor, cyclic guanosine monophosphate-adenosine monophosphate
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synthase, and inflammasomes.4,8-18 In this review, we discuss recent advances in the understanding
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of autophagy and inflammatory innate immune response, especially inflammasome activation.
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Identification and function of autophagy-related proteins
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Autophagic structures were first reported through electron microscopy studies in the 1950s and
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contributors to autophagosome formation were identified via yeast genetic screening in the
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1990s.19-22 Essential components of autophagosome formation are called as autophagy-related
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proteins (ATG proteins). At present, 38 ATG proteins have been identified in yeast. Core ATG
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proteins are phylogenetically highly conserved. Mammalian counterparts such as ULK1
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(mammalian ATG1), ATG3, ATG4s, ATG5, BECLIN1 (mammalian ATG6), ATG7, LC3A/B
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(mammalian ATG8), ATG9A, ATG10, ATG12, ATG13, ATG14, ATG16L1, FIP200 (mammalian
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ATG17), and WIPIs (mammalian ATG18) have been identified. Hence, autophagy is an
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evolutionarily conserved machinery for maintenance of cellular homeostasis. Core ATG proteins are
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classified into several functional units: the FIP200 complex; the class III PI3K complex; the
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ATG7-mediated, ubiquitin-like conjugation system; etc. (Figure 1). The coordinated action of
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functional ATG protein units induces the membrane trafficking events responsible for isolation
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membrane formation and subsequent autophagosome formation. Mice lacking ATG3, ATG5, ATG7,
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or ATG16L1 have been observed to die shortly after birth because of poor nutrition and energy
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depletion; therefore, autophagy-dependent maintenance of cellular homeostasis is required for
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survival. On the other hand, mice lacking BECLIN1 and FIP200 are embryonic lethal. Thus, ATG
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proteins have additional functions beyond simply inducing autophagosome formation.
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Selective clearance of damaged organelles and ubiquitinated proteins by autophagy
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Autophagy has been considered as a non-specific bulk degradation process. However, at present, it is
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clear that autophagy selectively eliminates damaged organelles, pathogen-containing vacuoles, and
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insoluble protein aggregates.18,23 The initial event that induces selective autophagy is ubiquitination
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of the substrate. Ubiquitination is a hallmark of unfavorable material within the cell and the
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autophagy system seeks to eliminate such material. The autophagosome engulfs damaged organelles
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and pathogen-containing vacuole that contain ubiquitinated membranes and ubiquitinated protein
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aggregates, for further degradation within the lysosome. Recent studies have identified p62, NDP52,
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and optineurin as the adaptor molecules that bind to ubiquitinated proteins and recruit ATG proteins
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to induce selective degradation of substrates by autophagy (Figure 2). As β-galactose chains
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normally exist in the lumen of endosomal and phagosomal compartments, exposure of β-galactose
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chains to cytosolic galectins is other hallmark of damaged endosomes and phagosomes. Adaptor
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protein, NDP52 binds to not only ubiquitin chains but also galectin-8, a cytosolic galectin, to recruit
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autophagy machinery toward the damaged endosome and phagosome. Thus, selective autophagy
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maintains cellular homeostasis by eliminating unfavorable material within the cell.
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The inflammasome, an innate immune structure, is mainly activated in myeloid cells such as
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macrophages and induces inflammation to protect the host from microbial infection.24,25 The
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inflammasome is a cytosolic protein complex and consists of PRRs such as NOD-like receptors
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(NLRs) and absent in melanoma 2 (AIM2), together with the adaptor protein, apoptosis-associated
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speck-like protein containing caspase recruitment domain (ASC), and the protease caspase-1.
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Activation of the inflammasome induces processing of pro-interleukin (IL)-1β and pro-IL-18 by
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caspase-1, which in turn induces production of these cytokines. Inflammasome-dependent cytokine
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production plays an important role in host defense against microbes. However, inflammasome is
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often wrongly activated by host-derived stimulatory factors and environmental irritants, and results
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in massive inflammation. Disruption of negative regulatory mechanisms and exposure to excess
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microbial components also cause massive inflammation, which in turn results in severe tissue
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damage, and subsequently the development of infectious and inflammatory disorders.
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ATG16L1 in lipopolysaccharide-induced inflammation
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Genome-wide association studies have revealed that single nucleotide polymorphisms of the gene
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encoding ATG16L1, an autophagy-related protein, is associated with susceptibility to Crohn's
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disease, a chronic inflammatory disease of the intestine.26-27 Mouse colitis models have confirmed
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that ATG16L1 is critical in maintenance of intestinal homeostasis and indicate that ATG16L1 is a
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genetic factor responsible for development of colitis. Hematopoietic cell-specific ATG16L1-deficient
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mice and mice expressing Crohn’s disease-associated ATG16L1 variant (T300A) are highly sensitive
171
to experimental colitis induced by dextran sulfate sodium (DSS) and pathogenic bacteria.11,28,29
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Macrophages lacking ATG16L1 and expressing Crohn’s disease-associated ATG16L1 variant
173
produce large quantities of IL-1β and IL-18 in response to lipopolysaccharide (LPS) (Figure 3).
174
Importantly, peripheral blood mononuclear cells from Crohn's disease patients expressing Crohn’s
175
disease-associated ATG16L1 variant also produce large quantities of IL-1β.28,29 Upon stimulation,
176
apoptotic proteases caspase-3 and -7 induce proteolytic cleavage of the Crohn’s disease-associated
177
ATG16L1 variant, resulting in destabilization of ATG16L1 (Figure 4).28,29 Thus, apoptotic caspases
178
enhance production of IL-1β in macrophages expressing Crohn’s disease-associated ATG16L1
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variant. Macrophages lacking ATG7 or treated with VPS34 inhibitor, 3-methyladenine produce large
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quantities of IL-1β in response to LPS, indicating that multiple autophagy-related proteins are
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involved in the response.11 Autophagy is therefore, involved in LPS-induced IL-1β production,
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though this is not a unique function of ATG16L1. On the other hand, ATG16L1 in intestinal
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epithelial cells is also important in the control of intestinal inflammation. Hypomorphic mutation of
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ATG16L1 gene in mice causes dysfunction of Paneth cells, a specialized epithelial cell, as well as
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elevated production of tumor necrosis factor and IFN-γ, resulting in enhanced susceptibility to
186
DSS-induced experimental colitis.30,31 Dysfunction of Paneth cells is also found in intestinal
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epithelial cell-specific, ATG5- and ATG7-deficient mice and is caused by defective host defense
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against norovirus. Thus, autophagy is involved in the prevention of aberrant inflammation and in
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maintenance of intestinal homeostasis.
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NLR family, pyrin domain containing 3 (NLRP3) is a well-characterized PRR belonging to the
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NOD-like receptor family.24,25 NLRP3 forms the inflammasome complex with adaptor protein, ASC
194
and
195
NLRP3-inflammasome is activated after exposure to various organelle stressors in myeloid cells
196
such as macrophages. Damage of phagosome-lysosome, mitochondria and endoplasmic reticulum
197
(ER) potently activates NLRP3-inflammasome and induces production of IL-1β and IL-18.32-36
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Reactive oxygen species (ROS) and mitochondrial DNA released from damaged mitochondria play a
199
key role in activation of the NLRP3-inflammasome.32-37 Phagosomal rupture and ER stress cause
200
mitochondrial damage. Adenosine triphosphate (ATP), a danger signal produced from dying cells,
201
causes mitochondrial damage to activate NLRP3-inflammasome, resulting in elimination of
202
unfavorable, accumulated, dead cells via inflammation. NLRP3-inflammasome detects phagosomal
203
rupture caused by bacterial lysins such as streptolysin and hemolysin, and induces inflammation to
204
protect the host from bacterial infection. NLRP3-inflammasome is wrongly activated by stimulatory
205
metabolites such as monosodium urate (MSU) crystals, cholesterol crystals, and palmitic acid, which
206
in turn induces severe tissue damage, resulting in development of metabolic diseases.
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NLRP3-inflammasome is also wrongly activated by environmental irritants such as asbestos and
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silica particles, leading to severe lung tissue damage.
induce maturation
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protease caspase-1
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Autophagy promotes turnover of organelles, thus preventing the accumulation of highly
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fragile, aging organelles as well as elimination of damaged organelles.1,2,18,23 Thus, autophagy
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suppresses organelle stress-induced activation of the NLRP3-inflammasome. As expected, in
212
autophagy-deficient macrophages, damaged mitochondria accumulate and produce excessive
213
amounts of ROS after stimulation by inducers such as MSU crystals, cholesterol crystals and
214
palmitic acid, resulting in the enhanced activation of the NLRP3-inflammasome (Figure 5).11,34,37-40
215
Accumulated
216
damaged
mitochondria
in
autophagy-deficient
mitochondrial DNA, a inducer of the NLRP3-inflammasome.
37
macrophages
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Adaptor protein p62 recognizes 6
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damaged mitochondria, where membrane proteins are ubiquitinated by PARKIN, and induces
218
selective autophagy.41 Amino acid sensor general control non-derepressible-2-dependent autophagy
219
in intestinal antigen-presenting cells suppresses inflammasome activation and is critical for
220
prevention of intestinal inflammation.42 During influenza A virus infection, nucleotide-binding
221
oligomerization domain containing protein 2 detects viral RNA and promotes autophagic elimination
222
of damaged mitochondria by inducing ULK1 phosphorylation and limits excessive activation of the
223
NLRP3-inflammasome
224
NLRP3-inflammasome-dependent inflammation and leads to exacerbation of various inflammatory
225
disorders such as sepsis, colitis, pneumonia, atherosclerosis, and diabetes.11,37-43 On the other hand,
226
inducers
227
NLRP3-inflammasome and alleviate inflammation-induced tissue damage.44-46
autophagy
suppress
6).43
Loss
production
of
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of
autophagy
IL-1β
and
IL-18
greatly
mediated
enhances
by
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(Figure
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Autophagy in non-canonical inflammasome activation
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Recent studies have identified that cytosolic LPS stimulates the non-canonical inflammasome to
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induce pyroptosis and production of IL-1α, IL-1β, and IL-18 in myeloid cells such as
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macrophages.25,47 Non-canonical inflammasome in mouse consists of caspase-11, whereas, in human,
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it consists of caspase-4 and -5. Although TLR4 is required for inducible expression of caspase-11 as
234
well as IL-1α and IL-1β, it is not involved in recognition of cytosolic LPS. During infection by
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gram-negative bacteria, guanylate-binding proteins are transcriptionally induced by TLRs and
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promote membrane rupture of the bacteria-containing phagosome.48 Membrane rupture of
237
bacteria-containing phagosome releases bacterial LPS into the cytosol, thereby facilitating detection
238
of LPS by the non-canonical inflammasome. Autophagy deficiency causes accumulation of the
239
damaged bacteria-containing phagosome and greatly enhances activation of the non-canonical
240
inflammasome. Sensor proteins such as NDP52 recognize ubiquitin chains and galectin-8 in
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damaged bacteria-containing phagosome, and induce selective autophagy. Thus, autophagy
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eliminates bacteria-containing phagosome and prevents excessive induction of non-canonical
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inflammasome-mediated inflammation (Figure 7).
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Autophagic degradation of ASC and IL-1β β
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Autophagy suppresses activation of the AIM2- and NLRP3-inflammasomes. Inducers of these
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inflammasomes cause lysine 63-linked ubiquitination of ASC, an adaptor molecule of
248
inflammasomes.49 The ubiquitin sensor p62 binds to ubiquitinated ASC and induces its degradation
249
by inducing selective autophagy, resulting in suppression of inflammasome activation (Figure 8).
250
Furthermore, the autophagosome engulfs IL-1β and delivers it into lysosomal compartments for
251
degradation (Figure 8).50 Hence, autophagy disrupts multiple steps of inflammasome activation to
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prevent excessive inflammation. 7
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253 Autophagy in IL-1β β secretion
255
Autophagy suppresses activation of the inflammasome under normal and nutrient-rich conditions.
256
However, a recent study has shown that autophagy is often involved in the unconventional secretion
257
of IL-1β under nutrient-starved conditions (Figure 9)51. Macrophages lacking ATG5 produce scarce
258
amounts of IL-1β in response to nigericin, an inducer of the NLRP3-inflammasome under
259
nutrient-starved conditions. Golgi reassembly stacking protein and RAB8A, membrane traffic
260
regulators, are also involved in nigericin-induced secretion of IL-1β. On the other hand, other studies
261
have shown that autophagy limits production of IL-1β under nutrient-starved conditions.49 At present,
262
differences in conditions that can account for this apparent discrepancy are not clarified. Hence,
263
further study is needed to define the role of autophagy in IL-1β production under nutrient-starved
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conditions.
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Recent studies have revealed the involvement of autophagy in inflammatory innate immune response.
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In myeloid cells such as macrophages, autophagy suppresses inflammasome activation by promoting
271
elimination of hazardous material such as damaged organelles.11,37-43,48 Autophagy deficiency in
272
myeloid cells causes aberrant activation of inflammasome, leading to development of inflammatory
273
disorders (Figure 10). Therefore, autophagy would be a promising therapeutic target for treatment of
274
inflammasome-related inflammatory disorders. Therapeutic agents capable of inducing autophagy
275
should be developed to alleviate inflammasome-dependent inflammation.
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Since the discovery of autophagy-related proteins, the role of autophagy in immune
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response has been extensively investigated.4,18 Autophagy is involved not only in PRR-mediated
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inflammation, but also in various immune responses such as pathogen elimination, antigen
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presentation, and immune cell development. However, the mechanism/s underlying autophagic
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control of the immune response are yet unclear. Future investigation will clarify the fundamental role
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of autophagy in immune response and provide a molecular basis for innovative drug development.
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Figure Legends
416
Fig.1. Autophagy.
417
The isolation membrane appears after exposure to stressors. Endoplasmic reticulum-mitochondria
418
contact site is a membrane source for the isolation membrane. The isolation membrane elongates
419
and engulfs cytosolic constituents to form the autophagosome, which fuses with lysosomes to form
420
the autolysosome, resulting in degradation of the engulfed constituents. Autophagy promotes the
421
reuse of cellular components, turnover of old organelles and elimination of unfavorable materials,
422
thus playing a vital role in the maintenance of cellular homeostasis and prevention of disease.
423
Autophagy-related proteins drive membrane trafficking necessary for the generation of isolation
424
membrane and autophagosomes. 11
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425 Fig.2. Autophagy eliminates damaged organelles.
427
Membranes of damaged phagosomes containing bacteria or crystals and damaged mitochondria are
428
ubiquitinated and targeted by adaptor proteins such as p62, NDP52 and optineurin. β-galactose
429
chains within the damaged phagosome are recognized by cytosolic galectin-8, which binds to
430
adaptor protein NDP52. Adaptor proteins recruit autophagy-related proteins to generate
431
autophagosomes in the proximity of the damaged organelle, which engulf and eliminate damaged
432
organelles after fusion with lysosomes.
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Fig.3. Autophagy suppresses TLR4-dependent production of IL-1β β and IL-18.
435
TLR4 detects LPS, triggers MyD88-dependent signaling pathway to activate the transcription factor,
436
nuclear factor-κB, and promotes pro-IL-1β expression. (A) TLR4 hardly induces production of
437
IL-1β and IL-18 in autophagy-competent normal macrophages, because autophagy suppresses
438
TRIF-dependent signaling pathway leading to ROS generation. (B) TRIF-mediated ROS production
439
is greatly enhanced in autophagy-deficient macrophages, which lack ATG16L1 expression. Thus,
440
autophagy-deficiency enhances LPS-induced production of IL-1β and IL-18.
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Fig.4. Crohn’s disease-associated ATG16L1 is susceptible to protease degradation.
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Crohn’s disease-associated ATG16L1 variant (T300A), but not wild-type ATG16L1, is processed by
444
caspase-3 and -7. When cells are exposed to severe stress, these caspases induce processing of
445
ATG16L1 (T300A) and make it unstable, resulting in loss of autophagy activity. Autophagy
446
deficiency causes massive inflammation leading to development of colitis.
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Fig.5. Autophagy suppresses NLRP3-inflammasome activation.
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Stimulatory particles such as MSU and cholesterol crystals cause phagosomal rupture, resulting in
450
mitochondrial damage. ATP released from dying cells stimulates P2X7R and causes mitochondrial
451
damage, which in turn releases ROS, causing activation of the NLRP3-inflammasome.
452
Mitochondrial damage also causes release of mitochondrial DNA (mtDNA) to activate the
453
NLRP3-inflmmasome. PARKIN, an E3 ubiquitin ligase, induces ubiquitination of a set of proteins
454
expressed on damaged mitochondria. Autophagy selectively eliminates damaged mitochondria after
455
sensing ubiquitinated proteins by adaptor protein p62. Autophagy suppresses activation of the
456
NLRP3-inflammasome by eliminating damaged mitochondria, which prevents excess production of
457
ROS and mtDNA.
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Fig.6. NOD2-dependent autophagy suppresses influenza virus-induced activation of the
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NLRP3-inflammasome. 12
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Influenza virus infection causes mitochondrial damage, causing ROS production, that results in
462
NLRP3-inflammasome activation. Autophagy eliminates damaged mitochondria to prevent excess
463
ROS production and subsequent activation of the NLRP3-inflammasome. However, influenza virus
464
also induces autophagy by stimulating NOD2. NOD2 deficiency causes impaired autophagy
465
induction, resulting in massive pulmonary inflammation during influenza virus infection.
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Fig.7. Autophagy suppresses non-canonical inflammasome activation.
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Gram-negative bacteria stimulate TLR4 via LPS and induce expression of guanylate-binding
469
proteins, caspase-11, pro-IL-1α and pro-IL-1β in mouse macrophages. Gram-negative bacteria also
470
induce phagosomal rupture, causing leakage of LPS into the cytosol, which then stimulates
471
caspase-11-driven non-canonical pathway of inflammasome activation. This leads to induction of
472
pyroptosis and production of IL-1α and IL-1β. Autophagy eliminates damaged phagosomes to
473
prevent aberrant activation of the NLRP3-inflammasome.
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Fig.9. Autophagy mediates IL-1β β secretion under starved condition.
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Under starved conditions, autophagy-related protein ATG5 is involved in secretion of IL-1β induced
482
by nigericin, an inducer of the NLRP3-inflammasome. Golgi reassembly stacking protein and
483
RAB8A are also involved in IL-1β secretion.
474
Fig.8. Autophagy suppresses inflammatory response by eliminating of ASC and IL-1β β.
476
Ubiquitinated ASC is targeted by adaptor proteins p62 and is recruited to the autophagosome. IL-1β
477
is detected in LC3-positive cytosolic compartment. Autophagosome engulfs these substrates and
478
eliminates them after fusion with lysosome.
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Fig.10. Autophagy deficiency causes inflammasome-related inflammatory disorders.
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Autophagy deficiency caused by gene disruption results in accumulation of damaged organelles,
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ASC, and IL-1β after exposure to various stressors such as microbes and stimulatory metabolites.
488
Thus, autophagy deficiency induces aberrant activation of the inflammasomes and subsequent
489
production of IL-1β and IL-18, leading to the development of inflammatory disorders.
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Lysosome Isolation membrane
Membrane source
Autophagosome Fusion
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• Ubiquitin-like conjugation system ATG7, ATG10, ATG12, ATG5, ATG16L1, ATG3, LC3A/B, ATG4s (mATG8) • PI3K complex VPS34, VPS15, BECLIN1, ATG14 (mATG6) • FIP200 complex FIP200, ATG13, ATG101, ULK1/2 (mATG17) (mATG1) • Others ATG9A, ATG2A/B, WIPIs (mATG18)
Roles Reuse of cellular components Promotion of cellular turnover Elimination of damaged organelle Elimination of invading pathogens
Immune response Host defense Cell death / differentiation Tumorigenesis Nutrient metabolism
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Autophagosome
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(B)
Bacteria
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LPS TLR4
MyD88
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MSU crystals Cholesterol crystals
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Adaptor protein p62
ACCEPTED MANUSCRIPT
Influenza virus
Mitochondrial damage
M AN U
SC
RI PT
NOD2
Inhibition
ROS production
TE D
EP
IL-18 production
Autophagy induction
AC C
NLRP3-inflammasome activation
ACCEPTED MANUSCRIPT
Bacteria
LPS TLR4
Ubiquitination
SC
Inhibition
M AN U
Ub Ub Ub
GBPs expression
LPS leakage
Autophagy induction
IL-1α and IL-1β production Pyroptosis
EP
TE D
Non-canonical inflammasome activation
AC C
Caspase-11, pro-IL-1α, pro-IL-1β expression
RI PT
Phagocytosis Phagosomal rupture
ACCEPTED MANUSCRIPT
Autophagosome
Ub Ub Ub
Engulfment
ASC ASC IL-1β
EP
TE D
M AN U
SC
IL-1β
IL-1β
Autophagyrelated proteins
AC C
Adaptor protein p62
Degradation
RI PT
Targeting
ASC
Ub Ub Ub Ub Ub Ub
ACCEPTED MANUSCRIPT
Autophagosome
IL-1β
IL-1β IL-1β
Autophagy -related proteins
Secretion IL-1β
M AN U TE D EP AC C
NLRP3-inflammasome activation by Nigericin
IL-1β
SC
IL-1β IL-1β
Engulfment
RI PT
Nutrient starvation
RAB8A GRASP
IL-1β
ACCEPTED MANUSCRIPT
Bacteria
Inflammatory response
Inflammatory disorders Colitis
Accumulation of damaged organelles and signal regulators
Sepsis
RI PT
Stimulation
Aberrant activation of inflammasome
Pneumonia
SC
Influenza virus
Severe damage of tissues
TE D
Diabetes
Autophagy-deficiency
EP
Fatty acid
Atherosclerosis
ATG5-, ATG7-, ATG16L1-deficient mice ATG16L1 T300A mice / T300A patients PARKIN-deficient mice P62-deficient mice
AC C
Cholesterol crystals
M AN U
Overproduction of IL-1β / IL-18