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Autophagy Accompanies Inflammasome Activation to Moderate Inflammation by Eliminating Active Inflammasomes
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16 Autophagy Accompanies Inflammasome Activation to Moderate Inflammation by Eliminating Active Inflammasomes Neel R. Nabar, Chong-Shan Shi and John H. Kehrl O U T L I N E Pharmacological Manipulation of Autophagy Regulates Inflammasome Activation 350 Autophagosomes Engulf Active Inflammasomes for Delivery to the Lysosome 351
Introduction 344 Inflammasome Components and Activation Inflammasome Components NLRP3 Activation AIM2 Activation
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Autophagy Function and Activation 348 Autophagy and Its Role in Immunity 348 Autophagosome Formation and Cargo Selection 348 Inflammasome Triggers Induce Autophagy 349 Poly(dA:dT) and Nigericin Induce Autophagy Independent of IL-1β 349 Inflammasome Sensor Activation Induces Nucleotide Exchange on RalB 350 Autophagy Regulates Inflammasome Activation 350 M.A. Hayat (ed): Autophagy, Volume 12. DOI: http://dx.doi.org/10.1016/B978-0-12-812146-7.00016-0
Connecting the Inflammasome and Autophagy Pathways Inflammasomes Recruit p62 for Delivery to Autophagosomes Inflammasomes Undergo K63-Linked Ubiquitination Upon Activation
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Autophagy Effects Inflammasome Activity in Primary Human Cells
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Conclusion and Perspective
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Acknowledgments 354 References 354
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© 2017, published by Elsevier Inc.
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Abstract
Inflammation is an important physiological response to noxious stimuli, but unchecked inflammation is pathophysiological and can result in tissue destruction and autoimmune disease. Inflammasomes are large multimeric signaling complexes of the innate immune system involved in the proteolytic cleavage and activation of interleukin-1 beta (IL-1β) and IL-18. Inflammasome signaling is a key early step in generating the inflammatory response as IL-1β is a critical cytokine in mediating systemic inflammation and has a variety of downstream effects. Aberrant inflammasome activation is the basis of many human autoinflammatory diseases, thus delineation of feedback loops regulating inflammasome signaling is of great importance to understanding autoinflammatory conditions. Autophagy is a conserved degradation pathway that delivers damaged organelles and aged protein complexes to the lysosome for destruction. It evolved both as a cellular recycling system and as a quality control system to ensure cellular homeostasis. This chapter covers the dynamic interplay between inflammasome activation and autophagy induction, with a specific focus on the cellular and molecular mechanisms by which autophagy feedback inhibits inflammasome signaling.
INTRODUCTION Inflammation is the initial physiological response to harmful stimuli and can be defined as the delivery of leukocytes and plasma proteins to the propagating site. During this process, inflammatory exudate containing host defense cells and proteins that normally reside in the blood are delivered to the extravascular site of infection (or injury) via the postcapillary venules (Medzhitov, 2008). Inflammation was first thought primarily to be a response to pathogenic invasion or acute injury, but it is now appreciated that a myriad of stimuli including dead cells, irritants, or persistent self-derived danger signals can initiate the inflammatory process (Janeway and Medzhitov, 2002). Controlled inflammation is crucial in maintaining organismal homeostasis in response to noxious stimuli, but unchecked inflammation can result in a pathological inflammatory state and autoinflammatory disease (Medzhitov, 2008). Propagation of the inflammatory response occurs through a complex cascade that amplifies the initial signal, and the cytokine interleukin-1 beta (IL-1β) is one of the key early systemic proinflammatory mediators during inflammation (Church et al., 2008). The IL-1 protein family has 11 members, of which IL-1α, IL-1β, IL-1 receptor antagonist, and IL-18 have been extensively studied. In humans, IL-1β is produced primarily by blood monocytes, tissue macrophages, and dendritic cells where it is translated as an inactive precursor (pro-IL-1β) ready for cleavage by caspase-1. After its cleavage, IL-1β undergoes noncanonical secretion from the cell to propagate the inflammatory response in both an autocrine and a paracrine manner (Dinarello, 2009). The IL-1β response is mediated by the IL-1 receptor (IL-1R) and increases the levels of blood neutrophils and many important immunomodulatory factors, such as adrenocorticotropic hormone, acute phase proteins, and several cytokines (including IL-6) (Roux-Lombard et al., 1992; Shieh et al., 1993; Fantuzzi and Dinarello, 1996). IL-1β also increases mesenchymal expression of intracellular adhesion molecule 1 and endothelial expression of vascular adhesion molecular 1, which in concert with increased cytokine levels is responsible for promoting the hallmark delivery of immunocompetent cells and proteins from the blood to the extravascular tissue during inflammation. Though IL-1β plays a critical role in physiological inflammation, excess IL-1β can be a causative factor of pathophysiological inflammation. Excess IL-1β in the bloodstream
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clinically manifests as fever, lowered pain threshold, vasodilation, and hypotension via IL-1R-dependent increases in nitric oxide and prostaglandin E2 (Molina-Holgado et al., 2000; Teng et al., 2002). Owing to the pleiotropic effects and clinical manifestations of excess IL-1β, IL-1β neutralization has now become a mainstay treatment for autoinflammatory diseases. Autoimmune diseases encompass all diseases with inappropriate immune activation, whereas autoinflammatory diseases are a subset of autoimmune diseases that are by definition IL-1β mediated and therefore respond to anti-IL-1β therapy. These diseases, including Familial Mediterranean Fever, Familial cold autoinflammatory syndrome, Muckle–Wells syndrome, and Neonatal-onset multiinflammatory disease, are characterized by periodicity, lack of association with class II MHC haplotypes, and strong association with exogenous triggers (Dinarello, 2009). Apart from their dependence on IL-1β, autoinflammatory diseases can be distinguished from autoimmune diseases in that the inappropriate immune activation is within the evolutionarily conserved innate arm of the immune system (Church et al., 2008). The initial signal in both physiological inflammation and pathological autoinflammatory diseases is generated by innate resident tissue macrophages that employ germline-encoded pattern recognition receptors (PRRs) to recognize critical and conserved pathogen-associated molecular patterns (PAMPs) or self-derived danger signals called damage-associated molecular patters (DAMPs). The most well-studied PRRs are the Toll-like receptors (TLRs), which recognize extracellular or endosome-localized PAMPs to initiate downstream signaling and activate NF-kappaB (NFκB) (Janeway and Medzhitov, 2002). NFκB activity is responsible for the transcriptional upregulation of pro-IL-1β and is required for the release of active IL-1β subsequent to cleavage and activation of the zymogen. Importantly the NFκB pathway has a critical negative feedback mechanism mediated by the ubiquitinating/deubiquitinating protein A20, which serves as a brake in TLR-mediated immune activation. Absence of A20 in mice leads to severe inflammation, cachexia, and early cell death, highlighting the importance of feedback mechanisms in preventing uncontrolled inflammation and disease (Lee et al., 2000). Studies into the mechanisms of autoinflammatory diseases lead to the discovery of a new class of PRRs, the nucleotide-oligomerization domain (NOD)-leucine rich repeat (LRR)containing receptors (NLRs) (Hoffman et al., 2001; Aksentijevich et al., 2002). Upon PAMP or DAMP recognition, these PRRs activate multimeric signaling complexes called inflammasomes that trigger procaspase-1 activation and the subsequent cleavage and secretion of IL-1β and IL-18 (Yang et al., 1998; Martinon et al., 2002). The strong association of inflammasome components with human autoinflammatory diseases underscores the importance of these signaling pathways in the innate immune response. This chapter will focus on the feedback mechanisms responsible for keeping inflammasome activation in check after initiation of these pathways. Autophagy, the cellular response to starvation and an essential lysosome-dependent degradation pathway responsible for clearing damaged organelles and aged proteins from the cell, has been implicated in the regulation of inflammasomes. Genetic deletion of the autophagy protein ATG16L makes normally poorly responsive macrophages sensitive to inflammasome activation induced by lipopolysaccharide (LPS) (Saitoh et al., 2008). Following this report and others, our group investigated the mechanisms through which autophagy limits inflammation and found autophagosomes directly engulf inflammasomes after their activation resulting in their degradation (Shi et al., 2012).
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This chapter will cover the current understanding of inflammasome activation as well as the detailed signaling mechanisms through which inflammasome stimuli activate autophagy. Finally, it will address the regulatory role of autophagy on inflammasome activation and its importance to immune homeostasis.
INFLAMMASOME COMPONENTS AND ACTIVATION Inflammasome Components All the major inflammasomes are organized similarly and are named according to the protein responsible for their scaffolding, which is typically the sensor molecule. Once an inflammasome sensor is stimulated by its trigger, the sensor molecule recruits the adaptor protein apoptosis-associated speck-like protein containing caspase activation and recruitment domain (CARD) (ASC) resulting in its oligomerization and the recruitment of procaspase-1. The ASC protein is encoded by the PYCARD gene and is a key component of all known inflammasomes. Structurally, it is a two-death fold motif containing protein with an amino-terminal pyrin domain (required for binding the sensor molecule) and a carboxyterminal CARD that mediates its caspase-1 interaction (Proell et al., 2013; Vajjhala et al., 2014). Upon binding to the sensor, ASC forms a large protein speck consisting of a multimer of ASC dimers that form via homotypic pyrin–pyrin interactions (Fernandes-Alnemri et al., 2007; Bryan et al., 2009). Formation of the ASC speck brings multiple procaspase-1 monomers into spacial proximity, allowing autoproteolytic caspase-1 activation and the formation of active heterotetrameric caspase-1. Cleaved caspase-1 then activates a variety of proteins, including pro-IL-1β and pro-IL-18 (Thornberry et al., 1992). Thus IL-1β release is regulated at two distinct steps—transcriptionally by the NFκB pathway and by posttranslational cleavage via the inflammasome pathways. The sensor molecule of each inflammasome is responsible for determining its specificity. Most inflammasome sensors are from the NLR family, including NLRP1 (NOD-, LRR-, and pyrin domain–containing 1), NLRP3, NLRP6, NLRP7, NLRP12, and NLRC4 (NOD-, LRR-, and CARD-containing 4). All of the NLRPs (except NLRP1) contain an amino-terminal pyrin domain that is important in mediating the interaction with ASC. NLRC4 contains an amino-terminal CARD domain allowing it to directly recruit and activate caspase-1, though interaction with ASC greatly augments NLRC4-dependent caspase-1 activation and is probably initially mediated via NLRC4/ASC CARD–CARD interactions. The sensor proteins all contain a centrally located NACHT domain with ATPase activity that is probably involved in protein oligomerization as well as carboxy-terminal LRRs that may be involved in ligand recognition (Latz et al., 2013). Despite the structural similarities between the NLR sensors, they are able to recognize a variety of stimuli. NLRP1 is activated by the anthrax lethal toxin, NLRP3 by a variety of toxic stimuli, and NLRC4 by bacterial flagellin. It is currently unclear whether NLRP6 and NLRP12 are able to form active inflammasomes, and other functions for these proteins have been described. Although most of the inflammasome sensors are of the NLR family, AIM2 and RIG-1 are non-NLR sensors capable of activating the inflammasome machinery (Fernandes-Alnemri et al., 2010; Rathinam et al., 2010; Latz et al., 2013). Activation of the NLRP3 and AIM2 inflammasomes is well studied and will be discussed in detail in the following section.
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NLRP3 Activation Activation of the NLRP3 inflammasome in primary macrophages requires two distinct steps. First, NLRP3 must be primed (e.g., signal I) by an upstream stimulus that induces NFκB signaling. In vitro, LPS stimulation of TLR4 is sufficient as it induces the transcriptional upregulation of NLRP3 (and pro-IL-1β) (Bauernfeind et al., 2009). Priming also rapidly licenses the NLRP3 signaling pathway via deubiquitination of NLRP3 and linear ubiquitination of ASC, both of which are required for NLRP3 inflammasome assembly (Juliana et al., 2012; Py et al., 2013; Rodgers et al., 2014). The NLRP3 inflammasome can be activated after priming (e.g., signal II) by a variety of stimuli, including extracellular ATP, the ionophore nigericin, uric acid crystal, alum salts, and streptolysin O. In the absence of signal II, pro-IL-1β remains upregulated but fails to be processed into its active form. As many distinct stimuli are capable of stimulating NLRP3, it is believed that these stimuli converge at a common downstream event that activates NLRP3. Potassium efflux from the cell, mitochondrial reactive oxygen species (ROS) generation, NLRP3 mitochondrial translocation, translocation of cardiolipin to the outer mitochondrial membrane, and release of cathepsins from the lysosome have all been proposed as the downstream unifying event. However, none of the identified downstream events were initially recognized to be induced by all NLRP3 agonists (Guo et al., 2015), and only after recent reexamination of NLRP3 stimuli, it has been established that potassium efflux is in fact a common downstream event (Broz and Dixit, 2016). However, it is still unknown as to whether NLRP3 directly senses the potassium decrease or if it senses some other phenomenon associated with potassium efflux. Nonetheless three independent reports determined that the small kinase NIMArelated kinase 7 (NEK7) acts downstream of potassium efflux and is required for NLRP3 activation (Schmid-Burgk et al., 2015; He et al., 2016; Shi et al., 2016). NEK7 plays an important role in mitotic cell progression, as it is required for the microtubule nucleation activity of the centrosome. After NLRP3 activation, NEK7 binds directly with NLRP3 and promotes inflammasome assembly in a kinase-independent manner.
AIM2 Activation Though the precise mechanisms mediating NLRP3 activation are still unclear, the events mediating AIM2 inflammasome assembly are better understood. AIM2 is a pyrin and HIN (hematopoietic expression, interferon-inducible nature, and nuclear localization) domain– containing family member that directly binds cytosolic DNA. The existence of a cytosolic DNA sensor was first proposed after the observation that microbial and host dsDNA induced caspase-1 activation through ASC but independent of NLRP3, TLR, or IFN signaling (Muruve et al., 2008). Shortly thereafter, multiple groups reported that AIM2 is the receptor mediating this response (Fernandes-Alnemri et al., 2009; Roberts et al., 2009) and that deficiency of AIM2 increased host susceptibility to DNA viruses and certain bacteria (Fernandes-Alnemri et al., 2010; Rathinam et al., 2010). Structurally, AIM2 exists in an autoinhibited state due to interactions between its pyrin domain (required for ASC recruitment) and its HIN DNA-binding domain (Jin et al., 2012). AIM2 requires a dsDNA strand of 80+ base pairs for optimal inflammasome activation, and DNA binding disrupts the autoinhibitory state of AIM2 freeing the pyrin domain to recruit and serve as a scaffold for ASC oligomerization (Jin et al., 2012).
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AUTOPHAGY FUNCTION AND ACTIVATION Autophagy and Its Role in Immunity Autophagy is a fundamental and conserved eukaryotic degradation pathway that delivers cellular organelles and large protein complexes to the lysosome for degradation. Autophagy evolved both as a cellular recycling and as a quality control mechanism. As such, it is constitutively active at low levels in most cells but can be potently induced by specific stimuli. Upon autophagy induction a membrane sac called an isolation membrane expands into a double-membrane vesicle termed the autophagosome. During this process, cytoplasmic constituents are either selectively or nonselectively enveloped by the autophagosome membrane and confined to the autophagosome lumen. The autophagosome eventually merges with a lysosome, forming an autophagolysosome and resulting in degradation of the sequestered material (Noda and Inagaki, 2015). Though autophagy was first recognized as a cellular response to nutrient deprivation, it is now clear that autophagy is induced by a variety of events causing cellular stress. One such event is pathogen invasion, and the induction of autophagy can help to clear intracellular protozoa, bacteria, and viruses by direct sequestration and delivery to the lysosome for destruction (Deretic et al., 2013). The importance of autophagy in the immune response is further highlighted by its function in antigen presentation (Schmid et al., 2007). A variety of immunologic stimuli can trigger autophagy, including LPS and IL-1β. LPS stimulation activates TLR4 and targets Beclin-1 (a key component of the Class III PI(3)K autophagosome initiation complex) to the TLR adaptor proteins MyD88 and TRIF, resulting in its TRAF6mediated K63-linked ubiquitination and activation (Shi and Kehrl, 2008, 2010). IL-1β is sufficient but not necessary for autophagy induction, and the molecular mechanisms driving IL-1β-mediated autophagy are not well understood.
Autophagosome Formation and Cargo Selection Autophagosome formation is an early event after autophagy induction and can be broken down into three steps: nucleation, expansion, and fusion of the isolation membrane. Genetic analyses have identified over 35 critical AuTophaGy (ATG) proteins in yeast and their mammalian counterparts. Each step in autophagosome formation requires the enzymatic activity of particular protein complexes containing ATG proteins. Specifically, nucleation relies on the ULK1 kinase induction complex consisting of ULK1/ATG13/FIP200, which is regulated via phosphorylation by the mammalian target of rapamycin (mTOR). This complex is important in the regulation of mAtg9 shuttling, which is thought to be important for the recruitment of autophagosomal membranes (Young et al., 2006; Kijanska et al., 2010; Kraft and Martens, 2012). Expansion relies on the Class III PI(3)K complex containing Beclin-1, ATG14L, VPS34, and VPS15. Nutrient deprivation regulates the activity of both these complexes as starvation inhibits mTOR to activate the ULK1 complex (Nazio et al., 2013) and releases Beclin-1 from Bcl-2-mediated inhibition to activate the Class III PI(3)K complex (Pattingre et al., 2005). The exocyst, a heterooctameric complex containing Sec3, Sec5, Sec6, Sec10, Sec15, Exo70, and Ex084, is also important in autophagy induction (Wu and Guo, 2015). After the
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recognition that the exocyst is a critical signaling scaffold in response to pathogens (Chien et al., 2006), the White Group followed up an earlier yeast two-hybrid screen using the exocyst component Sec3 as bait that identified both positive (FIP200 and Atg14L, members of the ULK1 and Class III PI(3)K complex, respectively) and negative (Rubicon) regulators of autophagy as interactors (Formstecher et al., 2005). They then looked at the small GTPases RalA and RalB responsible for mobilizing exocyst assembly to determine their role in autophagy. Bodemann et al. showed that RalB is both necessary and sufficient for the activation of autophagy. GTP-bound RalB, through direct binding with its effector Exo84, promotes the assembly of the ULK1 and Class III PI(3)K complexes on the exosome during starvation (Bodemann et al., 2011). Recent reports confirmed the importance of the exocyst in autophagy induction, but suggest a more complex tissue-specific situation (Tracy et al., 2016). After isolation membrane nucleation and expansion, fusion of its distal ends is required to complete autophagosome formation. An essential step in this process is the modification of LC3B-I to LC3B-II via its conjugation to phosphoethanolamine. Modification of LC3B tightly binds it to autophagosome membranes, making it a specific marker for autophagosomes and autophagolysosomes. The conversion of LC3B-I to LC3B-II also causes it to migrate more quickly on an electrophoretic gel, making immunoblot analysis of LC3B-I to LC3B-II conversion another useful assay to measure autophagic activity. For selective autophagy to occur, cargo must be delivered to the isolation membrane prior to its fusion. Certain adaptor proteins have evolved to allow for the delivery of protein complexes tagged for destruction to the autophagosome. p62, which was initially named sequestesome-1 due to its ability to form aggregates, is one such adaptor protein. In neurodegenerative diseases and proteinopathies of the liver, p62 has been shown to accumulate with ubiquitin-positive inclusions (Zatloukal et al., 2002; Kuusisto et al., 2008). p62 contains both an LC3-interacting region (LIR) motif and a C-terminal ubiquitin-associated domain (UBA) (Shin, 1998). The UBA domain of p62 binds mono- and polyubiquitinated proteins but has a preference for monoubiquitinated substrates in vitro (Long et al., 2008). In vivo, p62 oligomerization allows it to bind strongly to proteins with multiple ubiquitin tags, while its LIR domain allows for selective delivery to LC3 during autophagosome formation (Pankiv et al., 2007; Ichimura et al., 2008). After delivery to LC3 by p62-linked substrates the isolation membrane curves around the cargo prior to fusion (Wurzer et al., 2015).
INFLAMMASOME TRIGGERS INDUCE AUTOPHAGY Poly(dA:dT) and Nigericin Induce Autophagy Independent of IL-1β To investigate crosstalk between inflammasome activation and autophagy (Shi et al., 2012), our group first used the human monocyte cell line THP-1 stably expressing green flourescent protein (GFP) linked LC3 (GFP-LC3). Basally, GFP-LC3 has a diffuse cytoplasmic expression pattern but accumulates into green fluorescent dots upon autophagy activation. After differentiation of THP-1 GFP-LC3 cells into macrophages by treatment with phorbol myristate acetate (PMA), we transfected poly(dA:dT) to activate the AIM2 inflammasome. Poly(dA:dT) transfection resulted in an increased number of cells with LC3 dots and more
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LC3 dots per cell. This was further increased upon treatment with Ed64 and pepstatin A (lysosome inhibitors) and blocked by treatment with 3-methyladenine (3-MA, an inhibitor of autophagosome formation), confirming that the LC3 response was specific to autophagy. Because IL-1β is released with inflammasome activation and IL-1β autocrine signaling can induce autophagy, we added an IL-1β neutralizing antibody with poly(dA:dT) stimulation. The increase in LC3 dots was preserved, showing that the induction if autophagy is independent of IL-1β. Finally, we used siRNA knockdown of Beclin-1 and AIM2 to show that autophagosome formation was dependent on the sensor and autophagy machinery. To confirm these results in primary cells and test the pertinence of our findings in other inflammasomes, we assessed LC3B-I to LC3B-II conversion in bone marrow–derived macrophages after treatment with poly(dA:dT) or nigericin (an NLRP3 activator). WT macrophages showed increased LC3B-II after treatment with both poly(dA:dT) and nigericin, which was abrogated in poly(dA:dT)-treated AIM2−/− BMDMs. The increased LC3 conversion was conserved in ASC−/− and caspase-1−/− BMDMs, confirming that the induction of autophagy is dependent on the sensor but not on the inflammasome assembly.
Inflammasome Sensor Activation Induces Nucleotide Exchange on RalB The exchange of GDP for GTP on RalB leads to its binding to Exo84, allowing the exocyst to serve as a scaffold required for the formation of the isolation membrane (Bodemann et al., 2011). To assess whether inflammasome activators trigger RalB nucleotide exchange, we used a Ral-binding protein 1 precipitation assay to pull down GTP-bound RalB followed by immunoblotting for RalB. Treatment of WT BMDMs with poly(dA:dT), LPS + ATP, and uric acid crystals resulted in rapid RalB nucleotide exchange and the induction of autophagy. This effect was blunted in AIM2−/− BMDMs after poly(dA:dT) treatment, indicating that RalB nucleotide exchange was dependent on the sensor. Finally, siRNA knockdown of RalB reduced the induction of autophagy after poly(dA:dT) transfection. Together, these studies provided evidence that inflammasome activation induces autophagy by promoting nucleotide exchange on RalB.
AUTOPHAGY REGULATES INFLAMMASOME ACTIVATION Pharmacological Manipulation of Autophagy Regulates Inflammasome Activation To investigate the functional effects of autophagy on inflammasome activation, we examined AIM2 inflammasome activity after reducing autophagy with the Class III PI(3)K inhibitor 3-MA and enhancing autophagy by treatment with rapamycin (an mTOR inhibitor) or starvation. Using differentiated THP-1 cells, we activated the AIM2 inflammasome by poly(dA:dT) transfection and immunoblotted for cleaved caspase-1 (p20) and mature IL-1β in the cell lysate and supernatant. As expected, transfection of poly(dA:dT) resulted in more p20 and IL-1β in the lysate and supernatant compared to untransfected controls. Interestingly, reduction of autophagy with 3-MA resulted in more p20 and IL-1β in both the lysate and the supernatant, while starvation and rapamycin reduced p20 and IL-1β in both.
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To confirm that this phenomenon was preserved with NLRP3 activation, we performed the same experiment using uric acid and nigericin as stimulants. The use of PMA to differentiate THP-1 monocytes into macrophages stimulates some NFκB activity, which is sufficient for NLRP3 priming. Again, pharmacological inhibition of autophagy augmented the inflammasome activation, while enhancing autophagy decreased the inflammasome activation. Finally, as RalB is required for autophagy activation, we knocked down RalB with siRNA to see the effect on IL-1β release. As expected, deficiency of RalB resulted in increased IL-1β in the supernatant due to decreased autophagy. To determine if autophagy regulates inflammasome activation in response to bacterial infection, we infected mouse BMDMs with either live or dead Mycobacterium tuberculosis (TB). There is a complex interplay between autophagy, the inflammasome and TB, as autophagy inhibits TB survival in infected macrophages but TB also actively suppresses the inflammasome via its zinc-dependent metalloprotease Zmp1 (Gutierrez et al., 2004; Master et al., 2008). Nevertheless, both live and dead TB induced autophagy and promoted IL-1β secretion into the supernatant. Consistent with previous results, inhibition of autophagy with 3-MA resulted in increased inflammasome activation, suggesting that autophagy regulates inflammasome activation in response to a variety of stimuli.
Autophagosomes Engulf Active Inflammasomes for Delivery to the Lysosome After determining that autophagy regulates inflammasome activation, we used confocal microscopy to visualize a potential interaction between the inflammasomes and autophagosomes. As cytosolic DNA binds AIM2, we first used Cy3-tagged poly(dA:dT) as a surrogate marker for AIM2 after transfection into differentiated THP-1 GFP-LC3 cells. Using time lapse confocal microscopy in live cells, we clearly saw that GFP-LC3-labeled autophagosomes merge with cytosolic Cy3-poly(dA:dT). We then used immunostaining of ASC after transfection of unlabeled poly(dA:dT) in differentiated THP-1 GFP-LC3 cells and again saw partial colocalization between inflammasomes and autophagosomes. To determine whether NLRP3 inflammasomes also colocalize with autophagosomes, we treated differentiated THP-1 GFP-LC3 cells with LPS (for priming) and ATP (signal II) and immunostained for ASC. The use of surface-rendering software to process the z-section stack of images allowed us to detect ASC structures completely enclosed in LC3-positive structures resembling autophagosomes. Finally, electron microscopy of immunostained ASC in ultrathin slices of THP-1 cells after AIM2 or NLRP3 activation showed that some of the ASC immunostaining was located inside autophagosomes. These data clearly indicate that autophagosomes can engulf active inflammasomes. As autophagosome eventually merge with lysosomes, we immunostained for ASC and the lysosome marker LAMP-1 after poly(dA:dT) transfection and saw clear colocalization. Next, we verified that the sensors AIM2 and NLRP3 colocalized with GFP-LC3 and LAMP-1 after poly(dA:dT) and LPS + ATP stimulation, respectively. To verify our microscopic observations biochemically, we stimulated differentiated THP-1 cells with poly(dA:dT) and fractionated the cell lysate into inflammasome- and autophagosome-rich fractions. Stimulation with poly(dA:dT) increased AIM2 in both the autophagosome and inflammasome fractions. However, inhibition of autophagosome formation by 3-MA in poly(dA:dT)-transfected cells resulted in less AIM2 in the autophagosome fraction and more in the inflammasome fraction. Conversely, treatment with
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rapamycin or starvation to enhance autophagy increased AIM2 in the autophagosome fraction. These data, in concert with the confocal microscopy data and the data showing autophagy manipulation, regulate IL-1β and p20 production, strongly indicating that both AIM2 and NLRP3 inflammasomes can be engulfed by autophagosomes and delivered to lysosomes for destruction.
CONNECTING THE INFLAMMASOME AND AUTOPHAGY PATHWAYS Inflammasomes Recruit p62 for Delivery to Autophagosomes p62 is an adaptor protein that binds ubiquitinated substrates for delivery to the autophagosome. To determine if p62 had a role in inflammasome delivery to the autophagosome, we immunoprecipitated ASC and immunoblotted for p62 in unstimulated and poly(dA:dT)-treated THP-1 cells. These proteins interacted basally, indicating that autophagy may keep inadvertent inflammasome activation in check even in the absence of inflammasome stimulation. AIM2 activation by poly(dA:dT) increased the interaction between ASC and p62 as well as the interaction between AIM2 and p62. Imaging analysis was also consistent in that p62 colocalized with ASC after AIM2 inflammasome induction. Using LPS + ATP to stimulate the NLRP3 inflammasome, we detected more p62 and ASC interaction after NLRP3 activation. 3D rendering of confocal microscopy z-section stacks showed ASC aggregates studded with p62 after NLRP3 activation. Finally, to directly confirm that p62 plays a role in the delivery of inflammasomes to the autophagosome, we used siRNA knockdown of p62 followed by fractionation of cell lysates into inflammasome- and autophagosome-rich portions. Upon poly(dA:dT stimulation), knockdown of p62 resulted in increased AIM2 in the inflammasome fraction with a concomitant decrease of AIM2 in the autophagosome fraction. This was accompanied by an increase in caspase-1 activation and IL-1β release after inflammasome stimulation in p62-deficient cells. These experiments indicate that inflammasomes recruit p62, which helps deliver them to autophagosomes for eventually delivery and destruction by lysosomes.
Inflammasomes Undergo K63-Linked Ubiquitination Upon Activation As p62 binds ubiquitinated substrates, we set out to determine if inflammasomes are ubiquitinated upon activation to allow p62 targeting. Immunoprecipitation of ASC followed by immunoblotting with antibodies that recognize ubiquitin, K63-linked ubiquitin, and ASC all showed smeared patterns consistent with ubiquitination after poly(dA:dT) stimulation. As p62 has a preference for K63-linked ubiquitinated substrates, this result is not entirely unexpected (Seibenhener et al., 2004). Imaging analysis also showed strong colocalization after poly(dA:dT) stimulation and coimmunostaining for ASC and polyubiquitinated substrates, confirming that ASC is indeed ubiquitinated after AIM2 inflammasome activation. In sum, these results indicate that polyubiquitination of activated inflammasomes allows p62 targeting of inflammasomes for delivery to autophagosomes.
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A 2016 study identified a separate yet complementary mechanism by which AIM2 inflammasomes are delivered to the autolysosome for destruction (Liu et al., 2016). Upon dsDNA stimulation, tripartite motif 11 (TRIM11) binds AIM2 and undergoes auto-polyubiquitination, thereby facilitating the AIM2–p62 interaction and promoting AIM2 degradation by selective autophagy. Moreover, deficiency in TRIM11 resulted in excess AIM2 activation after stimulation with poly(dA:dT), further corroborating our observation that autophagy negatively regulates inflammasome activation.
AUTOPHAGY EFFECTS INFLAMMASOME ACTIVITY IN PRIMARY HUMAN CELLS To confirm that the autophagy regulates inflammasome activation in human primary cells, we used elutriated monocytes and stimulated with nigericin or poly(dA:dT). Both treatments increased LC3B-I to LC3B-II conversion, indicating that inflammasome activators were able to stimulate autophagy. Nigericin and poly(dA:dT) both increased cleaved caspase-1 and IL-1β in the supernatant, which was enhanced by treatment with 3-MA and reduced by treatment with rapamycin or starvation. These studies confirmed that autophagy is a conserved feedback mechanism, which is induced upon inflammasome activation and functions to limit IL-1β production and inflammatory signaling by targeting ubiquitinated inflammasomes for destruction.
CONCLUSION AND PERSPECTIVE Aberrant inflammasome activation has been implicated as a causative factor for many autoinflammatory diseases but has also been shown to potentiate the pathogenesis of a variety of diseases, including gout, atherosclerosis, Crohn’s disease, and lung damage following exposure to asbestos or silica (Saitoh and Akira, 2016). Our studies clearly show that autophagy directly limits the inflammatory response by engulfing inflammasomes (summarized in Fig. 16.1). The clinical importance of this is underscored by the fact that single nucleotide polymorphisms (SNPs) in ATG16L1 are associated with a susceptibility to Crohn’s disease (Hampe et al., 2007). Crohn’s disease causing ATG16L1 SNP results in inappropriate degradation of the protein and deficient autophagy, and PMBCs from patients with Crohn’s disease with this variant produce large quantities of IL-1β (Lassen et al., 2014; Murthy et al., 2014). There is also evidence that autophagy regulates inflammasome activation at other steps. Autophagosomes are able to directly engulf IL-1β and deliver it to the lysosome for degradation (Harris et al., 2011). Autophagy is also involved in the turnover of mitochondria, and autophagy-deficient macrophages have an accumulation of damaged mitochondria. This accumulation of damaged mitochondria increases ROS levels after NLRP3 stimulation by inducers, thereby increasing NLRP3 activation (Zhou et al., 2011). Moving forward, the effect of autophagy at different steps in inflammasome activation needs to be further investigated. However, modulation of autophagy may be a viable therapeutic target for inflammasome-related inflammatory disorders in the future.
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FIGURE 16.1 Autophagy accompanies inflammasome activation to limit inflammation. The feedback loop by which autophagy regulates inflammasome activation is summarized. Detection of its ligand by an inflammasome sensor molecule causes RalB nucleotide exchange, allowing Exo84 exocyst assembly and autophagy induction. Concomitantly, inflammasome assembly, activation of caspase-1, and ASC K63-linked ubiquitination follow. Ubiquitination of ASC results in the recruitment of p62, which delivers active inflammasomes to the autophagosome for engulfment and eventual destruction via delivery to the lysosome.
Acknowledgments This research was supported by the Intramural Research Program of NIAID, NIH. We thank Dr. Anthony Fauci for his continued support.
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Introduction 344 Inflammasome Components and Activation Inflammasome Components NLRP3 Activation AIM2 Activation
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Autophagy Function and Activation 348 Autophagy and Its Role in Immunity 348 Autophagosome Formation and Cargo Selection 348 Inflammasome Triggers Induce Autophagy 349 Poly(dA:dT) and Nigericin Induce Autophagy Independent of IL-1β 349 Inflammasome Sensor Activation Induces Nucleotide Exchange on RalB 350 Autophagy Regulates Inflammasome Activation 350 M.A. Hayat (ed): Autophagy, Volume 12. DOI: http://dx.doi.org/10.1016/B978-0-12-812146-7.00016-0
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Abstract
Inflammation is an important physiological response to noxious stimuli, but unchecked inflammation is pathophysiological and can result in tissue destruction and autoimmune disease. Inflammasomes are large multimeric signaling complexes of the innate immune system involved in the proteolytic cleavage and activation of interleukin-1 beta (IL-1β) and IL-18. Inflammasome signaling is a key early step in generating the inflammatory response as IL-1β is a critical cytokine in mediating systemic inflammation and has a variety of downstream effects. Aberrant inflammasome activation is the basis of many human autoinflammatory diseases, thus delineation of feedback loops regulating inflammasome signaling is of great importance to understanding autoinflammatory conditions. Autophagy is a conserved degradation pathway that delivers damaged organelles and aged protein complexes to the lysosome for destruction. It evolved both as a cellular recycling system and as a quality control system to ensure cellular homeostasis. This chapter covers the dynamic interplay between inflammasome activation and autophagy induction, with a specific focus on the cellular and molecular mechanisms by which autophagy feedback inhibits inflammasome signaling.
INTRODUCTION Inflammation is the initial physiological response to harmful stimuli and can be defined as the delivery of leukocytes and plasma proteins to the propagating site. During this process, inflammatory exudate containing host defense cells and proteins that normally reside in the blood are delivered to the extravascular site of infection (or injury) via the postcapillary venules (Medzhitov, 2008). Inflammation was first thought primarily to be a response to pathogenic invasion or acute injury, but it is now appreciated that a myriad of stimuli including dead cells, irritants, or persistent self-derived danger signals can initiate the inflammatory process (Janeway and Medzhitov, 2002). Controlled inflammation is crucial in maintaining organismal homeostasis in response to noxious stimuli, but unchecked inflammation can result in a pathological inflammatory state and autoinflammatory disease (Medzhitov, 2008). Propagation of the inflammatory response occurs through a complex cascade that amplifies the initial signal, and the cytokine interleukin-1 beta (IL-1β) is one of the key early systemic proinflammatory mediators during inflammation (Church et al., 2008). The IL-1 protein family has 11 members, of which IL-1α, IL-1β, IL-1 receptor antagonist, and IL-18 have been extensively studied. In humans, IL-1β is produced primarily by blood monocytes, tissue macrophages, and dendritic cells where it is translated as an inactive precursor (pro-IL-1β) ready for cleavage by caspase-1. After its cleavage, IL-1β undergoes noncanonical secretion from the cell to propagate the inflammatory response in both an autocrine and a paracrine manner (Dinarello, 2009). The IL-1β response is mediated by the IL-1 receptor (IL-1R) and increases the levels of blood neutrophils and many important immunomodulatory factors, such as adrenocorticotropic hormone, acute phase proteins, and several cytokines (including IL-6) (Roux-Lombard et al., 1992; Shieh et al., 1993; Fantuzzi and Dinarello, 1996). IL-1β also increases mesenchymal expression of intracellular adhesion molecule 1 and endothelial expression of vascular adhesion molecular 1, which in concert with increased cytokine levels is responsible for promoting the hallmark delivery of immunocompetent cells and proteins from the blood to the extravascular tissue during inflammation. Though IL-1β plays a critical role in physiological inflammation, excess IL-1β can be a causative factor of pathophysiological inflammation. Excess IL-1β in the bloodstream
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clinically manifests as fever, lowered pain threshold, vasodilation, and hypotension via IL-1R-dependent increases in nitric oxide and prostaglandin E2 (Molina-Holgado et al., 2000; Teng et al., 2002). Owing to the pleiotropic effects and clinical manifestations of excess IL-1β, IL-1β neutralization has now become a mainstay treatment for autoinflammatory diseases. Autoimmune diseases encompass all diseases with inappropriate immune activation, whereas autoinflammatory diseases are a subset of autoimmune diseases that are by definition IL-1β mediated and therefore respond to anti-IL-1β therapy. These diseases, including Familial Mediterranean Fever, Familial cold autoinflammatory syndrome, Muckle–Wells syndrome, and Neonatal-onset multiinflammatory disease, are characterized by periodicity, lack of association with class II MHC haplotypes, and strong association with exogenous triggers (Dinarello, 2009). Apart from their dependence on IL-1β, autoinflammatory diseases can be distinguished from autoimmune diseases in that the inappropriate immune activation is within the evolutionarily conserved innate arm of the immune system (Church et al., 2008). The initial signal in both physiological inflammation and pathological autoinflammatory diseases is generated by innate resident tissue macrophages that employ germline-encoded pattern recognition receptors (PRRs) to recognize critical and conserved pathogen-associated molecular patterns (PAMPs) or self-derived danger signals called damage-associated molecular patters (DAMPs). The most well-studied PRRs are the Toll-like receptors (TLRs), which recognize extracellular or endosome-localized PAMPs to initiate downstream signaling and activate NF-kappaB (NFκB) (Janeway and Medzhitov, 2002). NFκB activity is responsible for the transcriptional upregulation of pro-IL-1β and is required for the release of active IL-1β subsequent to cleavage and activation of the zymogen. Importantly the NFκB pathway has a critical negative feedback mechanism mediated by the ubiquitinating/deubiquitinating protein A20, which serves as a brake in TLR-mediated immune activation. Absence of A20 in mice leads to severe inflammation, cachexia, and early cell death, highlighting the importance of feedback mechanisms in preventing uncontrolled inflammation and disease (Lee et al., 2000). Studies into the mechanisms of autoinflammatory diseases lead to the discovery of a new class of PRRs, the nucleotide-oligomerization domain (NOD)-leucine rich repeat (LRR)containing receptors (NLRs) (Hoffman et al., 2001; Aksentijevich et al., 2002). Upon PAMP or DAMP recognition, these PRRs activate multimeric signaling complexes called inflammasomes that trigger procaspase-1 activation and the subsequent cleavage and secretion of IL-1β and IL-18 (Yang et al., 1998; Martinon et al., 2002). The strong association of inflammasome components with human autoinflammatory diseases underscores the importance of these signaling pathways in the innate immune response. This chapter will focus on the feedback mechanisms responsible for keeping inflammasome activation in check after initiation of these pathways. Autophagy, the cellular response to starvation and an essential lysosome-dependent degradation pathway responsible for clearing damaged organelles and aged proteins from the cell, has been implicated in the regulation of inflammasomes. Genetic deletion of the autophagy protein ATG16L makes normally poorly responsive macrophages sensitive to inflammasome activation induced by lipopolysaccharide (LPS) (Saitoh et al., 2008). Following this report and others, our group investigated the mechanisms through which autophagy limits inflammation and found autophagosomes directly engulf inflammasomes after their activation resulting in their degradation (Shi et al., 2012).
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This chapter will cover the current understanding of inflammasome activation as well as the detailed signaling mechanisms through which inflammasome stimuli activate autophagy. Finally, it will address the regulatory role of autophagy on inflammasome activation and its importance to immune homeostasis.
INFLAMMASOME COMPONENTS AND ACTIVATION Inflammasome Components All the major inflammasomes are organized similarly and are named according to the protein responsible for their scaffolding, which is typically the sensor molecule. Once an inflammasome sensor is stimulated by its trigger, the sensor molecule recruits the adaptor protein apoptosis-associated speck-like protein containing caspase activation and recruitment domain (CARD) (ASC) resulting in its oligomerization and the recruitment of procaspase-1. The ASC protein is encoded by the PYCARD gene and is a key component of all known inflammasomes. Structurally, it is a two-death fold motif containing protein with an amino-terminal pyrin domain (required for binding the sensor molecule) and a carboxyterminal CARD that mediates its caspase-1 interaction (Proell et al., 2013; Vajjhala et al., 2014). Upon binding to the sensor, ASC forms a large protein speck consisting of a multimer of ASC dimers that form via homotypic pyrin–pyrin interactions (Fernandes-Alnemri et al., 2007; Bryan et al., 2009). Formation of the ASC speck brings multiple procaspase-1 monomers into spacial proximity, allowing autoproteolytic caspase-1 activation and the formation of active heterotetrameric caspase-1. Cleaved caspase-1 then activates a variety of proteins, including pro-IL-1β and pro-IL-18 (Thornberry et al., 1992). Thus IL-1β release is regulated at two distinct steps—transcriptionally by the NFκB pathway and by posttranslational cleavage via the inflammasome pathways. The sensor molecule of each inflammasome is responsible for determining its specificity. Most inflammasome sensors are from the NLR family, including NLRP1 (NOD-, LRR-, and pyrin domain–containing 1), NLRP3, NLRP6, NLRP7, NLRP12, and NLRC4 (NOD-, LRR-, and CARD-containing 4). All of the NLRPs (except NLRP1) contain an amino-terminal pyrin domain that is important in mediating the interaction with ASC. NLRC4 contains an amino-terminal CARD domain allowing it to directly recruit and activate caspase-1, though interaction with ASC greatly augments NLRC4-dependent caspase-1 activation and is probably initially mediated via NLRC4/ASC CARD–CARD interactions. The sensor proteins all contain a centrally located NACHT domain with ATPase activity that is probably involved in protein oligomerization as well as carboxy-terminal LRRs that may be involved in ligand recognition (Latz et al., 2013). Despite the structural similarities between the NLR sensors, they are able to recognize a variety of stimuli. NLRP1 is activated by the anthrax lethal toxin, NLRP3 by a variety of toxic stimuli, and NLRC4 by bacterial flagellin. It is currently unclear whether NLRP6 and NLRP12 are able to form active inflammasomes, and other functions for these proteins have been described. Although most of the inflammasome sensors are of the NLR family, AIM2 and RIG-1 are non-NLR sensors capable of activating the inflammasome machinery (Fernandes-Alnemri et al., 2010; Rathinam et al., 2010; Latz et al., 2013). Activation of the NLRP3 and AIM2 inflammasomes is well studied and will be discussed in detail in the following section.
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NLRP3 Activation Activation of the NLRP3 inflammasome in primary macrophages requires two distinct steps. First, NLRP3 must be primed (e.g., signal I) by an upstream stimulus that induces NFκB signaling. In vitro, LPS stimulation of TLR4 is sufficient as it induces the transcriptional upregulation of NLRP3 (and pro-IL-1β) (Bauernfeind et al., 2009). Priming also rapidly licenses the NLRP3 signaling pathway via deubiquitination of NLRP3 and linear ubiquitination of ASC, both of which are required for NLRP3 inflammasome assembly (Juliana et al., 2012; Py et al., 2013; Rodgers et al., 2014). The NLRP3 inflammasome can be activated after priming (e.g., signal II) by a variety of stimuli, including extracellular ATP, the ionophore nigericin, uric acid crystal, alum salts, and streptolysin O. In the absence of signal II, pro-IL-1β remains upregulated but fails to be processed into its active form. As many distinct stimuli are capable of stimulating NLRP3, it is believed that these stimuli converge at a common downstream event that activates NLRP3. Potassium efflux from the cell, mitochondrial reactive oxygen species (ROS) generation, NLRP3 mitochondrial translocation, translocation of cardiolipin to the outer mitochondrial membrane, and release of cathepsins from the lysosome have all been proposed as the downstream unifying event. However, none of the identified downstream events were initially recognized to be induced by all NLRP3 agonists (Guo et al., 2015), and only after recent reexamination of NLRP3 stimuli, it has been established that potassium efflux is in fact a common downstream event (Broz and Dixit, 2016). However, it is still unknown as to whether NLRP3 directly senses the potassium decrease or if it senses some other phenomenon associated with potassium efflux. Nonetheless three independent reports determined that the small kinase NIMArelated kinase 7 (NEK7) acts downstream of potassium efflux and is required for NLRP3 activation (Schmid-Burgk et al., 2015; He et al., 2016; Shi et al., 2016). NEK7 plays an important role in mitotic cell progression, as it is required for the microtubule nucleation activity of the centrosome. After NLRP3 activation, NEK7 binds directly with NLRP3 and promotes inflammasome assembly in a kinase-independent manner.
AIM2 Activation Though the precise mechanisms mediating NLRP3 activation are still unclear, the events mediating AIM2 inflammasome assembly are better understood. AIM2 is a pyrin and HIN (hematopoietic expression, interferon-inducible nature, and nuclear localization) domain– containing family member that directly binds cytosolic DNA. The existence of a cytosolic DNA sensor was first proposed after the observation that microbial and host dsDNA induced caspase-1 activation through ASC but independent of NLRP3, TLR, or IFN signaling (Muruve et al., 2008). Shortly thereafter, multiple groups reported that AIM2 is the receptor mediating this response (Fernandes-Alnemri et al., 2009; Roberts et al., 2009) and that deficiency of AIM2 increased host susceptibility to DNA viruses and certain bacteria (Fernandes-Alnemri et al., 2010; Rathinam et al., 2010). Structurally, AIM2 exists in an autoinhibited state due to interactions between its pyrin domain (required for ASC recruitment) and its HIN DNA-binding domain (Jin et al., 2012). AIM2 requires a dsDNA strand of 80+ base pairs for optimal inflammasome activation, and DNA binding disrupts the autoinhibitory state of AIM2 freeing the pyrin domain to recruit and serve as a scaffold for ASC oligomerization (Jin et al., 2012).
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AUTOPHAGY FUNCTION AND ACTIVATION Autophagy and Its Role in Immunity Autophagy is a fundamental and conserved eukaryotic degradation pathway that delivers cellular organelles and large protein complexes to the lysosome for degradation. Autophagy evolved both as a cellular recycling and as a quality control mechanism. As such, it is constitutively active at low levels in most cells but can be potently induced by specific stimuli. Upon autophagy induction a membrane sac called an isolation membrane expands into a double-membrane vesicle termed the autophagosome. During this process, cytoplasmic constituents are either selectively or nonselectively enveloped by the autophagosome membrane and confined to the autophagosome lumen. The autophagosome eventually merges with a lysosome, forming an autophagolysosome and resulting in degradation of the sequestered material (Noda and Inagaki, 2015). Though autophagy was first recognized as a cellular response to nutrient deprivation, it is now clear that autophagy is induced by a variety of events causing cellular stress. One such event is pathogen invasion, and the induction of autophagy can help to clear intracellular protozoa, bacteria, and viruses by direct sequestration and delivery to the lysosome for destruction (Deretic et al., 2013). The importance of autophagy in the immune response is further highlighted by its function in antigen presentation (Schmid et al., 2007). A variety of immunologic stimuli can trigger autophagy, including LPS and IL-1β. LPS stimulation activates TLR4 and targets Beclin-1 (a key component of the Class III PI(3)K autophagosome initiation complex) to the TLR adaptor proteins MyD88 and TRIF, resulting in its TRAF6mediated K63-linked ubiquitination and activation (Shi and Kehrl, 2008, 2010). IL-1β is sufficient but not necessary for autophagy induction, and the molecular mechanisms driving IL-1β-mediated autophagy are not well understood.
Autophagosome Formation and Cargo Selection Autophagosome formation is an early event after autophagy induction and can be broken down into three steps: nucleation, expansion, and fusion of the isolation membrane. Genetic analyses have identified over 35 critical AuTophaGy (ATG) proteins in yeast and their mammalian counterparts. Each step in autophagosome formation requires the enzymatic activity of particular protein complexes containing ATG proteins. Specifically, nucleation relies on the ULK1 kinase induction complex consisting of ULK1/ATG13/FIP200, which is regulated via phosphorylation by the mammalian target of rapamycin (mTOR). This complex is important in the regulation of mAtg9 shuttling, which is thought to be important for the recruitment of autophagosomal membranes (Young et al., 2006; Kijanska et al., 2010; Kraft and Martens, 2012). Expansion relies on the Class III PI(3)K complex containing Beclin-1, ATG14L, VPS34, and VPS15. Nutrient deprivation regulates the activity of both these complexes as starvation inhibits mTOR to activate the ULK1 complex (Nazio et al., 2013) and releases Beclin-1 from Bcl-2-mediated inhibition to activate the Class III PI(3)K complex (Pattingre et al., 2005). The exocyst, a heterooctameric complex containing Sec3, Sec5, Sec6, Sec10, Sec15, Exo70, and Ex084, is also important in autophagy induction (Wu and Guo, 2015). After the
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recognition that the exocyst is a critical signaling scaffold in response to pathogens (Chien et al., 2006), the White Group followed up an earlier yeast two-hybrid screen using the exocyst component Sec3 as bait that identified both positive (FIP200 and Atg14L, members of the ULK1 and Class III PI(3)K complex, respectively) and negative (Rubicon) regulators of autophagy as interactors (Formstecher et al., 2005). They then looked at the small GTPases RalA and RalB responsible for mobilizing exocyst assembly to determine their role in autophagy. Bodemann et al. showed that RalB is both necessary and sufficient for the activation of autophagy. GTP-bound RalB, through direct binding with its effector Exo84, promotes the assembly of the ULK1 and Class III PI(3)K complexes on the exosome during starvation (Bodemann et al., 2011). Recent reports confirmed the importance of the exocyst in autophagy induction, but suggest a more complex tissue-specific situation (Tracy et al., 2016). After isolation membrane nucleation and expansion, fusion of its distal ends is required to complete autophagosome formation. An essential step in this process is the modification of LC3B-I to LC3B-II via its conjugation to phosphoethanolamine. Modification of LC3B tightly binds it to autophagosome membranes, making it a specific marker for autophagosomes and autophagolysosomes. The conversion of LC3B-I to LC3B-II also causes it to migrate more quickly on an electrophoretic gel, making immunoblot analysis of LC3B-I to LC3B-II conversion another useful assay to measure autophagic activity. For selective autophagy to occur, cargo must be delivered to the isolation membrane prior to its fusion. Certain adaptor proteins have evolved to allow for the delivery of protein complexes tagged for destruction to the autophagosome. p62, which was initially named sequestesome-1 due to its ability to form aggregates, is one such adaptor protein. In neurodegenerative diseases and proteinopathies of the liver, p62 has been shown to accumulate with ubiquitin-positive inclusions (Zatloukal et al., 2002; Kuusisto et al., 2008). p62 contains both an LC3-interacting region (LIR) motif and a C-terminal ubiquitin-associated domain (UBA) (Shin, 1998). The UBA domain of p62 binds mono- and polyubiquitinated proteins but has a preference for monoubiquitinated substrates in vitro (Long et al., 2008). In vivo, p62 oligomerization allows it to bind strongly to proteins with multiple ubiquitin tags, while its LIR domain allows for selective delivery to LC3 during autophagosome formation (Pankiv et al., 2007; Ichimura et al., 2008). After delivery to LC3 by p62-linked substrates the isolation membrane curves around the cargo prior to fusion (Wurzer et al., 2015).
INFLAMMASOME TRIGGERS INDUCE AUTOPHAGY Poly(dA:dT) and Nigericin Induce Autophagy Independent of IL-1β To investigate crosstalk between inflammasome activation and autophagy (Shi et al., 2012), our group first used the human monocyte cell line THP-1 stably expressing green flourescent protein (GFP) linked LC3 (GFP-LC3). Basally, GFP-LC3 has a diffuse cytoplasmic expression pattern but accumulates into green fluorescent dots upon autophagy activation. After differentiation of THP-1 GFP-LC3 cells into macrophages by treatment with phorbol myristate acetate (PMA), we transfected poly(dA:dT) to activate the AIM2 inflammasome. Poly(dA:dT) transfection resulted in an increased number of cells with LC3 dots and more
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LC3 dots per cell. This was further increased upon treatment with Ed64 and pepstatin A (lysosome inhibitors) and blocked by treatment with 3-methyladenine (3-MA, an inhibitor of autophagosome formation), confirming that the LC3 response was specific to autophagy. Because IL-1β is released with inflammasome activation and IL-1β autocrine signaling can induce autophagy, we added an IL-1β neutralizing antibody with poly(dA:dT) stimulation. The increase in LC3 dots was preserved, showing that the induction if autophagy is independent of IL-1β. Finally, we used siRNA knockdown of Beclin-1 and AIM2 to show that autophagosome formation was dependent on the sensor and autophagy machinery. To confirm these results in primary cells and test the pertinence of our findings in other inflammasomes, we assessed LC3B-I to LC3B-II conversion in bone marrow–derived macrophages after treatment with poly(dA:dT) or nigericin (an NLRP3 activator). WT macrophages showed increased LC3B-II after treatment with both poly(dA:dT) and nigericin, which was abrogated in poly(dA:dT)-treated AIM2−/− BMDMs. The increased LC3 conversion was conserved in ASC−/− and caspase-1−/− BMDMs, confirming that the induction of autophagy is dependent on the sensor but not on the inflammasome assembly.
Inflammasome Sensor Activation Induces Nucleotide Exchange on RalB The exchange of GDP for GTP on RalB leads to its binding to Exo84, allowing the exocyst to serve as a scaffold required for the formation of the isolation membrane (Bodemann et al., 2011). To assess whether inflammasome activators trigger RalB nucleotide exchange, we used a Ral-binding protein 1 precipitation assay to pull down GTP-bound RalB followed by immunoblotting for RalB. Treatment of WT BMDMs with poly(dA:dT), LPS + ATP, and uric acid crystals resulted in rapid RalB nucleotide exchange and the induction of autophagy. This effect was blunted in AIM2−/− BMDMs after poly(dA:dT) treatment, indicating that RalB nucleotide exchange was dependent on the sensor. Finally, siRNA knockdown of RalB reduced the induction of autophagy after poly(dA:dT) transfection. Together, these studies provided evidence that inflammasome activation induces autophagy by promoting nucleotide exchange on RalB.
AUTOPHAGY REGULATES INFLAMMASOME ACTIVATION Pharmacological Manipulation of Autophagy Regulates Inflammasome Activation To investigate the functional effects of autophagy on inflammasome activation, we examined AIM2 inflammasome activity after reducing autophagy with the Class III PI(3)K inhibitor 3-MA and enhancing autophagy by treatment with rapamycin (an mTOR inhibitor) or starvation. Using differentiated THP-1 cells, we activated the AIM2 inflammasome by poly(dA:dT) transfection and immunoblotted for cleaved caspase-1 (p20) and mature IL-1β in the cell lysate and supernatant. As expected, transfection of poly(dA:dT) resulted in more p20 and IL-1β in the lysate and supernatant compared to untransfected controls. Interestingly, reduction of autophagy with 3-MA resulted in more p20 and IL-1β in both the lysate and the supernatant, while starvation and rapamycin reduced p20 and IL-1β in both.
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To confirm that this phenomenon was preserved with NLRP3 activation, we performed the same experiment using uric acid and nigericin as stimulants. The use of PMA to differentiate THP-1 monocytes into macrophages stimulates some NFκB activity, which is sufficient for NLRP3 priming. Again, pharmacological inhibition of autophagy augmented the inflammasome activation, while enhancing autophagy decreased the inflammasome activation. Finally, as RalB is required for autophagy activation, we knocked down RalB with siRNA to see the effect on IL-1β release. As expected, deficiency of RalB resulted in increased IL-1β in the supernatant due to decreased autophagy. To determine if autophagy regulates inflammasome activation in response to bacterial infection, we infected mouse BMDMs with either live or dead Mycobacterium tuberculosis (TB). There is a complex interplay between autophagy, the inflammasome and TB, as autophagy inhibits TB survival in infected macrophages but TB also actively suppresses the inflammasome via its zinc-dependent metalloprotease Zmp1 (Gutierrez et al., 2004; Master et al., 2008). Nevertheless, both live and dead TB induced autophagy and promoted IL-1β secretion into the supernatant. Consistent with previous results, inhibition of autophagy with 3-MA resulted in increased inflammasome activation, suggesting that autophagy regulates inflammasome activation in response to a variety of stimuli.
Autophagosomes Engulf Active Inflammasomes for Delivery to the Lysosome After determining that autophagy regulates inflammasome activation, we used confocal microscopy to visualize a potential interaction between the inflammasomes and autophagosomes. As cytosolic DNA binds AIM2, we first used Cy3-tagged poly(dA:dT) as a surrogate marker for AIM2 after transfection into differentiated THP-1 GFP-LC3 cells. Using time lapse confocal microscopy in live cells, we clearly saw that GFP-LC3-labeled autophagosomes merge with cytosolic Cy3-poly(dA:dT). We then used immunostaining of ASC after transfection of unlabeled poly(dA:dT) in differentiated THP-1 GFP-LC3 cells and again saw partial colocalization between inflammasomes and autophagosomes. To determine whether NLRP3 inflammasomes also colocalize with autophagosomes, we treated differentiated THP-1 GFP-LC3 cells with LPS (for priming) and ATP (signal II) and immunostained for ASC. The use of surface-rendering software to process the z-section stack of images allowed us to detect ASC structures completely enclosed in LC3-positive structures resembling autophagosomes. Finally, electron microscopy of immunostained ASC in ultrathin slices of THP-1 cells after AIM2 or NLRP3 activation showed that some of the ASC immunostaining was located inside autophagosomes. These data clearly indicate that autophagosomes can engulf active inflammasomes. As autophagosome eventually merge with lysosomes, we immunostained for ASC and the lysosome marker LAMP-1 after poly(dA:dT) transfection and saw clear colocalization. Next, we verified that the sensors AIM2 and NLRP3 colocalized with GFP-LC3 and LAMP-1 after poly(dA:dT) and LPS + ATP stimulation, respectively. To verify our microscopic observations biochemically, we stimulated differentiated THP-1 cells with poly(dA:dT) and fractionated the cell lysate into inflammasome- and autophagosome-rich fractions. Stimulation with poly(dA:dT) increased AIM2 in both the autophagosome and inflammasome fractions. However, inhibition of autophagosome formation by 3-MA in poly(dA:dT)-transfected cells resulted in less AIM2 in the autophagosome fraction and more in the inflammasome fraction. Conversely, treatment with
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rapamycin or starvation to enhance autophagy increased AIM2 in the autophagosome fraction. These data, in concert with the confocal microscopy data and the data showing autophagy manipulation, regulate IL-1β and p20 production, strongly indicating that both AIM2 and NLRP3 inflammasomes can be engulfed by autophagosomes and delivered to lysosomes for destruction.
CONNECTING THE INFLAMMASOME AND AUTOPHAGY PATHWAYS Inflammasomes Recruit p62 for Delivery to Autophagosomes p62 is an adaptor protein that binds ubiquitinated substrates for delivery to the autophagosome. To determine if p62 had a role in inflammasome delivery to the autophagosome, we immunoprecipitated ASC and immunoblotted for p62 in unstimulated and poly(dA:dT)-treated THP-1 cells. These proteins interacted basally, indicating that autophagy may keep inadvertent inflammasome activation in check even in the absence of inflammasome stimulation. AIM2 activation by poly(dA:dT) increased the interaction between ASC and p62 as well as the interaction between AIM2 and p62. Imaging analysis was also consistent in that p62 colocalized with ASC after AIM2 inflammasome induction. Using LPS + ATP to stimulate the NLRP3 inflammasome, we detected more p62 and ASC interaction after NLRP3 activation. 3D rendering of confocal microscopy z-section stacks showed ASC aggregates studded with p62 after NLRP3 activation. Finally, to directly confirm that p62 plays a role in the delivery of inflammasomes to the autophagosome, we used siRNA knockdown of p62 followed by fractionation of cell lysates into inflammasome- and autophagosome-rich portions. Upon poly(dA:dT stimulation), knockdown of p62 resulted in increased AIM2 in the inflammasome fraction with a concomitant decrease of AIM2 in the autophagosome fraction. This was accompanied by an increase in caspase-1 activation and IL-1β release after inflammasome stimulation in p62-deficient cells. These experiments indicate that inflammasomes recruit p62, which helps deliver them to autophagosomes for eventually delivery and destruction by lysosomes.
Inflammasomes Undergo K63-Linked Ubiquitination Upon Activation As p62 binds ubiquitinated substrates, we set out to determine if inflammasomes are ubiquitinated upon activation to allow p62 targeting. Immunoprecipitation of ASC followed by immunoblotting with antibodies that recognize ubiquitin, K63-linked ubiquitin, and ASC all showed smeared patterns consistent with ubiquitination after poly(dA:dT) stimulation. As p62 has a preference for K63-linked ubiquitinated substrates, this result is not entirely unexpected (Seibenhener et al., 2004). Imaging analysis also showed strong colocalization after poly(dA:dT) stimulation and coimmunostaining for ASC and polyubiquitinated substrates, confirming that ASC is indeed ubiquitinated after AIM2 inflammasome activation. In sum, these results indicate that polyubiquitination of activated inflammasomes allows p62 targeting of inflammasomes for delivery to autophagosomes.
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A 2016 study identified a separate yet complementary mechanism by which AIM2 inflammasomes are delivered to the autolysosome for destruction (Liu et al., 2016). Upon dsDNA stimulation, tripartite motif 11 (TRIM11) binds AIM2 and undergoes auto-polyubiquitination, thereby facilitating the AIM2–p62 interaction and promoting AIM2 degradation by selective autophagy. Moreover, deficiency in TRIM11 resulted in excess AIM2 activation after stimulation with poly(dA:dT), further corroborating our observation that autophagy negatively regulates inflammasome activation.
AUTOPHAGY EFFECTS INFLAMMASOME ACTIVITY IN PRIMARY HUMAN CELLS To confirm that the autophagy regulates inflammasome activation in human primary cells, we used elutriated monocytes and stimulated with nigericin or poly(dA:dT). Both treatments increased LC3B-I to LC3B-II conversion, indicating that inflammasome activators were able to stimulate autophagy. Nigericin and poly(dA:dT) both increased cleaved caspase-1 and IL-1β in the supernatant, which was enhanced by treatment with 3-MA and reduced by treatment with rapamycin or starvation. These studies confirmed that autophagy is a conserved feedback mechanism, which is induced upon inflammasome activation and functions to limit IL-1β production and inflammatory signaling by targeting ubiquitinated inflammasomes for destruction.
CONCLUSION AND PERSPECTIVE Aberrant inflammasome activation has been implicated as a causative factor for many autoinflammatory diseases but has also been shown to potentiate the pathogenesis of a variety of diseases, including gout, atherosclerosis, Crohn’s disease, and lung damage following exposure to asbestos or silica (Saitoh and Akira, 2016). Our studies clearly show that autophagy directly limits the inflammatory response by engulfing inflammasomes (summarized in Fig. 16.1). The clinical importance of this is underscored by the fact that single nucleotide polymorphisms (SNPs) in ATG16L1 are associated with a susceptibility to Crohn’s disease (Hampe et al., 2007). Crohn’s disease causing ATG16L1 SNP results in inappropriate degradation of the protein and deficient autophagy, and PMBCs from patients with Crohn’s disease with this variant produce large quantities of IL-1β (Lassen et al., 2014; Murthy et al., 2014). There is also evidence that autophagy regulates inflammasome activation at other steps. Autophagosomes are able to directly engulf IL-1β and deliver it to the lysosome for degradation (Harris et al., 2011). Autophagy is also involved in the turnover of mitochondria, and autophagy-deficient macrophages have an accumulation of damaged mitochondria. This accumulation of damaged mitochondria increases ROS levels after NLRP3 stimulation by inducers, thereby increasing NLRP3 activation (Zhou et al., 2011). Moving forward, the effect of autophagy at different steps in inflammasome activation needs to be further investigated. However, modulation of autophagy may be a viable therapeutic target for inflammasome-related inflammatory disorders in the future.
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FIGURE 16.1 Autophagy accompanies inflammasome activation to limit inflammation. The feedback loop by which autophagy regulates inflammasome activation is summarized. Detection of its ligand by an inflammasome sensor molecule causes RalB nucleotide exchange, allowing Exo84 exocyst assembly and autophagy induction. Concomitantly, inflammasome assembly, activation of caspase-1, and ASC K63-linked ubiquitination follow. Ubiquitination of ASC results in the recruitment of p62, which delivers active inflammasomes to the autophagosome for engulfment and eventual destruction via delivery to the lysosome.
Acknowledgments This research was supported by the Intramural Research Program of NIAID, NIH. We thank Dr. Anthony Fauci for his continued support.
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AUTOPHAGY