Autophagy and Inflammasome Interplay

Autophagy and Inflammation at a Glance

Autophagy is an intracellular process responsible for the turnover and recycling of proteins, as well as the clearance of damaged organelles and intracellular pathogens by means of lysosomal digestion. There are four types of autophagy: macroautophagy, microautophagy, chaperon-mediated autophagy, and noncanonical autophagy. Usually, macroautophagy is broadly referred to as autophagy. The hallmark of autophagy is the formation of double-membrane structures called autophagosomes (or autophagic vacuoles), which involves specialized proteins named autophagy-related proteins (ATGs) (Deretic et al., 2013). Part of the cytosol and its contents are enclosed by the autophagosomes, and for degradation to occur through lysosomal acidic hydrolases, autophagosomes fuse with lysosomes, thus forming the autophagolysosomes (Deretic et al., 2013; Arroyo et al., 2014; Chuang et al., 2014).

Basal levels of autophagy normally occur to preserve cell homeostasis, but its activation can also be triggered/exacerbated by cell stressors, such as endoplasmic reticulum (ER) and oxidative stress, mitochondrial damage, immune cell activation, and infection or nutrient deprivation. These noxious stimuli lead to mammalian target of rapamycin inhibition, with a consequent initiation of a cascade of events crucial to autophagosome formation and maturation (Levine et al., 2011; Arroyo et al., 2014; Chuang et al., 2014).

The central role of autophagy to cell homeostasis is highlighted by the close association of autophagy dysfunction with several debilitating conditions, such as cancer, neurodegenerative disorders, aging, and autoimmune and inflammatory diseases (Arroyo et al., 2014). Indeed, it has been demonstrated that autophagy is genetically related to immune cell responses and inflammation pathways (Deretic et al., 2013). Innate immunity, through promotion of inflammation, is the primary arm of the immune system, fighting invading pathogens or managing cell damage. Although inflammation is a central protective response, maintenance of a proinflammatory status might have dramatic deleterious effects in cells and tissues (O'Byrne and Dalgleish, 2001). In such a stressed environment, the autophagic pathway can be activated to prevent excessive inflammation (Jo et al., 2012).

Autophagy and immune/inflammatory interactions are regulated in a reciprocal way: autophagy proteins are able to evoke or inhibit immune or inflammatory responses, and immune- or inflammatory-related signals might trigger or suppress autophagy. Besides the genetic links between autophagy and immune disorders (e.g., autophagy-related genes polymorphism found in Crohn's disease), several evidences corroborate such interactions. The recognition of pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) by pattern recognition receptors has been shown to activate intracellular signaling cascades that cross talk and trigger autophagy (Deretic et al., 2013). Moreover, during inflammation, proinflammatory molecules are matured in specialized protein complexes, the inflammasomes, which are, in part, regulated by autophagic machinery.

Recently, autophagy was shown to control endogenous inflammasome activators, as well as inflammasome components and pro-interleukin (IL)-1β, thus regulating the secretion levels of proinflammatory cytokines, IL-1β and IL-18, and preventing an exacerbated inflammation (Levine et al., 2011; Deretic et al., 2013; Arroyo et al., 2014). The relationship between autophagy and inflammasomes in the context of inflammation will be further discussed in the following sections.

Inflammasome: Overview, Structure, and Signaling

After the discovery of the transmembrane toll-like receptors (TLRs), two new classes of microbial molecules cytosolic sensors were described: nod-like receptors (NLRs) recognizing bacterial peptidoglycans and RIG-I-like receptors (RLR) sensing viral RNA (Mogensen, 2009). NLRs also recognize endogenous molecules and sense cellular stresses, thus playing crucial roles in numerous aspects of immune and inflammatory responses, ranging from antimicrobial mechanisms to control of adaptive responses (Tsuchiya and Hara, 2014).

Humans and mice possess 23 and 34 reported NLRs, respectively. Most NLRs consist of three domains: a C-terminal leucine-rich repeat domain, a central nucleotide-binding oligomerization (NOD or NACHT) domain, and a variable N-terminal effector domain containing a caspase activation and recruitment domain (CARD), pyrin domain (PYD), or baculovirus inhibitor of apoptosis repeat domain (Franchi et al., 2012).

NLR recognition of PAMPs and DAMPs in the cytosol leads to the assembly of a signaling multimeric protein complex called the inflammasome. Besides the core component sensor molecule NLR, the inflammasome platform also integrates the adaptor apoptosis-associated speck-like protein containing a CARD (ASC) and caspase-1.

A wide range of substances that emerge during infections, tissue damage, or metabolic imbalances trigger the assembly of the inflammasome, thus initiating self-cleavage of procaspase-1 and the formation of the active heterotetrameric caspase-1. Active caspase-1 proteolytically cleaves the presynthesized pro-IL-1β and pro-IL-18 that are then secreted from the cell. In addition to the production of the proinflammatory cytokines, IL-1β and IL-18, the inflammasome/caspase-1 complex causes a rapid proinflammatory form of cell death called pyroptosis (Deretic, 2012). Several inflammasomes have been described so far, and among them, the best studied are the ones that contain NLRP3 (formerly known as Nalp3) or NLRC4 (formerly known as IPAF). Other NLR include NLRP1 (Nalp1), NLRP7 (NOD12), NLRP12 (monarch-1), and absent in melanoma 2, AIM2 (PYHIN).

The NLRP3 inflammasome is involved in many complicated multigenic diseases and metabolic disorders, including type 2 diabetes, gout, obesity, and atherosclerosis (Menu and Vince, 2011; Yuk and Jo, 2013). It has been shown that the NLRP3 inflammasome is triggered by a large variety of activators that do not share any structural similarities, such as pathogens (e.g., muramyl dipeptide moiety of peptidoglycans, lipopolysaccharides (LPS), Listeria monocytogenes, Candida albicans, Salmonella typhimurium, and influenza virus, among others) and endogenous sterile DAMP signals, including potassium efflux, amyloid deposits, monosodium urate, cholesterol crystals, mitochondrial reactive oxygen species (mtROS), and fatty acids (Davis et al., 2011).

A two-signal hypothesis was proposed to explain NLRP3 inflammasome activation responses (Yuk and Jo, 2013). The first signal, a priming signal, is provided by the TLR signaling pathway activation in a nuclear factor kappa light-chain-enhancer of activated B cells (NF-κB)-dependent manner and is a prerequisite for inflammasome activation through the induction of NLRP3 expression. Additionally, the first signal also triggers the synthesis of precursor forms of proinflammatory cytokines, including pro-IL-1β.

The second signal activates assembly of the NLRP3 inflammasome directly, thus inducing caspase-1 to cleave pro-IL-1β, and is triggered by potassium efflux, reactive oxygen species (ROS), or lysosomal proteases. Potassium efflux through membrane pores is triggered by ATP binding to the P²X₇ receptor, which results in the NLRP3 inflammasome assembly responses (Yuk and Jo, 2013; Tsuchiya and Hara, 2014). Another proposed mechanism suggests the activation of NLRP3 by cathepsin B released from damaged lysosomes following the frustrated phagocytosis of particulate matter, namely monosodium urate, silica, asbestos, amyloid deposits, and aluminum salts (Franchi et al., 2009).

mtROS were suggested to be an upstream activator of the NLRP3 inflammasome. Indeed, mitochondrial dysfunction concomitantly with loss of mitochondrial membrane potential and release of mitochondrial DNA (mtDNA) produces mtROS, which in turn activates the inflammasome through mitogen-activated protein kinases (MAPK), such as extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) (Harijith et al., 2014). However, as recently suggested, mitochondrial injury is not a requisite for NLRP3 inflammasome activation (Allam et al., 2014). Importantly, this genetic-based study revealed a nonapoptotic function of caspase-8 in inflammasome and proinflammatory cytokine level regulation.

Regarding what was described above, it seems that inflammasome activity is controlled at multiple levels and that several requirements need to be achieved to prompt the assembly.

The Role of Autophagy in Inflammasome Assembly and Activation

Several lines of evidence strongly suggest that NLRP3 inflammasome activity and subsequent excessive inflammation are negatively regulated by autophagy. For instance, mice that are deficient in Atg 16-like 1 (ATG16L1)—an essential component of the autophagic machinery—show higher IL-1β levels in response to stimulation (Saitoh et al., 2008). Although the mechanisms by which autophagy regulates inflammasome activation are still under debate, two hypothesis have been pointed out.

The first hypothesis suggests that inflammasomes or pro-IL-1β molecules are degraded by autophagosomes through the selective autophagic cargo receptor p62 (Harris et al., 2011; Shi et al., 2012). Accordingly, it was previously demonstrated that the induction of AIM2 or NLRP3 inflammasomes in macrophages triggered activation of the G protein RalB and autophagosome formation. After assembly, inflammasomes are ubiquitinated and recruit the autophagic adaptor p62, which assists their delivery to autophagosomes (Shi et al., 2012).

Recently, Harris et al. (2011) described a novel role for autophagy in regulating the production of IL-1β in antigen-presenting cells. After treatment of macrophages with TLRs ligands, pro-IL-1β was specifically sequestered into autophagosomes, whereas further autophagy activation with rapamycin induced the degradation of pro-IL-1β through its targeting for lysosomal degradation. Moreover, in vivo induction of autophagy with rapamycin reduced serum levels of IL-1β in mice challenged with LPS (Harris et al., 2011). The second hypothesis or additional mechanism, through which autophagy regulates the inflammasome, is related to autophagy removal of damaged mitochondria, preventing the release of mtROS and mtDNA into the cytoplasm and thereby limiting NLRP3 inflammasome assembly. In other words, autophagy controls inflammation by removing endogenous inflammasome-triggering DAMPs. Corroborating this hypothesis, the work of Nakahira et al. (2011) clearly demonstrated that depletion of the autophagic proteins, LC3B and beclin 1, enhances the activation of caspase-1 and secretion of IL-1β and IL-18 in macrophages. Furthermore, the depletion of autophagic proteins promoted the accumulation of dysfunctional mitochondria and cytosolic translocation of mtDNA in response to LPS and ATP. Finally, LC3B-deficient mice were shown to produce more caspase-1-dependent cytokines in two sepsis models and were susceptible to LPS-induced mortality. The authors concluded that autophagic proteins regulate NLRP3-dependent inflammation by preserving mitochondrial integrity (Nakahira et al., 2011).

Recently, a potential role for ER stress in innate immunity by activation of the NLRP3/caspase-1 inflammasome and involving the thioredoxin-interacting protein was described (Liu et al., 2015). Interestingly, resting NLRP3 localizes to ER structures, whereas upon inflammasome activation, both NLRP3 and its adaptor ASC are redistributed around the perinuclear space where they colocalize with ER and mitochondria organelle clusters. These observations suggest that the NLRP3 inflammasome senses mitochondrial/ER dysfunction and might explain the frequent association of mitochondrial/ER damage with inflammatory diseases (Zhou et al., 2011).

Van Der Burgh et al. (2014) demonstrated that hypersecretion of IL-1β and IL-18 requires ROS and is associated with an oxidized redox status of human monocytes. The IL-1β hypersecretion by monocytes was shown to involve a decrease in mitochondrial stability, release of mitochondrial content into the cytosol, and attenuation of autophagosomal degradation. Defective autophagy, established by Atg7 knockdown cells, resulted in prolonged cytosolic retention of damaged mitochondria and increased IL-1β secretion. The authors accomplish that autophagy dysfunction can prime monocytes for mitochondria-mediated NLRP3 inflammasome activation, thereby contributing to hypersecretion of IL-1β (Van Der Burgh et al., 2014).

An association between damaged mtDNA, autophagy, and NLRP3 inflammasome activation in macrophages, perhaps as a cellular self-protection mechanism, was also recently disclosed by Ding et al. (2014). In the referred work, treatment of macrophages with ROS inhibitors resulted in a markedly reduced expression of the NLRP3 inflammasome. To test a direct role of autophagy in this process, macrophages were treated with the autophagy inhibitor, 3-methyladenine (3-MA), or the inducer, rapamycin. Treatment with 3-MA resulted in the induction of NLRP3 expression, whereas rapamycin caused the inhibition of the NLRP3 inflammasome. The authors confirmed, that way, the role of mtDNA damage as a mediator of ROS generation, autophagy signals, and NLRP3 inflammasome activation (Ding et al., 2014). Furthermore, TLR2 or TLR4 engagement in macrophages induced plasminogen activator inhibitor type 2 expression (PAI-2), a serine protease inhibitor, which subsequently stabilized the autophagic protein beclin 1 to promote autophagy, resulting in decreased mtROS, NLRP3 protein levels, and pro-IL-1β processing (Chuang et al., 2013).

Reinforcing the modulatory role of autophagy in inflammasome activation through the clearance of damaged organelles, it was recently reported that the small-molecule andrographolide significantly attenuated in vivo colitis progression and tumor burden through inhibition of the NLRP3 inflammasome (Guo et al., 2014). Andrographolide was found to trigger mitochondrial autophagy (mitophagy) in macrophages, leading to an attenuation of the mitochondrial membrane potential collapse, which in turn inactivated the NLRP3 inflammasome. Finally, andrographolide-driven inhibition of the NLRP3 inflammasome and amelioration of murine models for colitis were significantly blocked by beclin1 knockdown or through various autophagy inhibitors (Guo et al., 2014). Accordingly, the deficiency of either autophagy or p62 in primary macrophages and in mice in vivo triggers inflammasome hyperactivation in response to LPS and ATP. Importantly, in these experiments, induction of protein misfolding resulted in more pronounced inflammasome activation in autophagy- or p62-deficient macrophages.

The accumulation of misfolded proteins can cause inflammasome activation by inducing generation of non-mtROS and lysosomal damage, leading to release of cathepsin B (Shin et al., 2013). Accordingly, inhibition of proteasomal degradation and autophagy in human retinal pigment epithelial cells results in increased amounts of intracellular protein aggregates and oxidative stress, which in turn upregulate the NLRP3 receptor and trigger caspase-1 activation, leading to an elevated production of biologically active IL-1β (Piippo et al., 2014). The study of the involvement of autophagy in inflammasome activation has been recently extended to neurodegenerative disorders. Currently, it is acknowledged that accumulation of β-amyloids and resultant inflammation are critical pathological features of Alzheimer's disease. Additionally, it is well established that loss of basal autophagy causes neurodegeneration. Cho et al. (2014) demonstrated that autophagy degrades extracellular β-amyloid fibrils and regulates the NLRP3 inflammasome in microglia.

Overall, these results indicate that autophagy accompanies inflammasome activation to alleviate inflammation by eliminating active inflammasomes or through removing endogenous inflammasome agonists. It is reasonable to conclude that altered proteostasis results in inflammasome activation, hence providing mechanisms for the association of altered proteostasis with inflammatory disorders, including autoinflammatory diseases, Crohn's disease, type 2 diabetes, atherosclerosis, cancer, cystic fibrosis, and ultimately aging (Deretic, 2012; Razani et al., 2012; Salminen et al., 2013; Wang et al., 2013; Saxena and Yeretssian, 2014).

Autophagy Modulation by Inflammasome

The regulation of inflammasomes by autophagy is counterbalanced by NLR-mediated control of autophagy. For instance, high-mobility group box 1 (HMGB1), a prototype DAMP, initially induces AIM2-dependent inflammasome activation and promotes a rapid IL-1β release. Subsequently, the HMGB1-DNA complex stimulates ATG5-dependent autophagy, which is paralleled by a cessation of AIM2 inflammasome activation and IL-1β release (Liu et al., 2014). This negative counter-regulatory mechanism of autophagy upon inflammasome activation was also disclosed in murine tricultures of neurons, astrocytes, and microglia treated with various inflammatory stimuli (i.e., Aβ42, LPS, or cytokines), revealing a major role of IL-1β in microglia–autophagy activation and supporting the close relationship between inflammation and autophagy (François et al., 2013).

Sun et al. (2013) addressed the role of caspase-1 in hepatocytes, nonimmune cells that express and activate the inflammasome, but do not produce significant amounts of IL-1β/IL-18. The authors demonstrated that caspase-1 activation prevents hepatocyte death after redox stress induced by hypoxia/reoxygenation. Mechanistically, caspase-1 reduced mitochondrial respiration and ROS by increasing mitophagy and subsequent clearance of mitochondria through the upregulation of the autophagy initiator, beclin 1 (Sun et al., 2013). Furthermore, NOD2, another member of the NLR family, was shown to play a protective role against virally triggered immunopathology through inhibition of the NLRP3 inflammasome and IL-18 production in a process dependent of mitophagy (Lupfer et al., 2013).

An exciting and emerging concept in the innate immunity field is the recognition that macrophages enlist autophagy to protect their cytoplasm from infection. Accordingly, Suzuki and Núñez (2008) demonstrated that Shigella infection induces caspase-1 activation and IL-1β processing in macrophages through NLRC4 and ASC assembly. Notably, infection also induced autophagy in an NLRC4-dependent way, which was associated with transient resistance to pyroptosis, providing a novel function for NLR proteins in bacterial–host interactions. In accordance, Byrne et al. (2013) tested the hypothesis that NLR proteins and caspase-1 also coordinate autophagy as a barrier to cytosolic infection. Exploiting classical bacterial and mouse genetics and kinetic assays of autophagy, the authors demonstrated that upon cytosolic contamination, primary mouse macrophages rely on the NLR proteins, NAIP5 and NLRC4, and on (pro-) caspase-1 protein to mount a rapid autophagic response that neutralizes proinflammatory cell death (Byrne et al., 2013).

We may therefore conclude that inflammasome components also regulate autophagy. Indeed, autophagy may function as a negative counter-regulatory mechanism for inflammasome activation, thus providing a checkpoint to limit the development of inflammation.

Cross Talk Between Autophagy, Inflammasome, and Protein Secretion

Besides autodigestive processes, in yeast, autophagy has additional roles, namely biogenesis functions (Reggiori and Klionsky, 2013). Recently, equivalent biosynthetic roles have been described for autophagy in mammals. Dupont et al. (2011) disclosed the contribution of autophagy to the unconventional secretory pathway for extracellular delivery of IL-1β. This export pathway depends on Atg5, inflammasome, and at least one of the two mammalian Golgi reassembly stacking protein (GRASP) paralogues, GRASP55 (GORASP2) and Rab8a (Dupont et al., 2011). More recently, it was demonstrated in a nasopharyngeal carcinoma and monocytic cell lines that end-binding protein 1 (EB1) is required for the autophagy-dependent secretion of IL-1β by AIM2 inflammasomes (Wang et al., 2014). Furthermore, Ohman et al. (2014) demonstrated, in human macrophages, that the dectin-1/Syk pathway activates an unconventional, vesicle-mediated protein secretion that is dependent both on the inflammasome and autophagy activity.

The active involvement of autophagy in unconventional secretion, for instance, through exosomes, expands the functional manifestations of autophagy beyond autodigestive and quality control roles in mammals. It enables a subset of cytosolic proteins devoided of signal peptide sequences, and thus unable to access the conventional pathway through the ER, to enter in an autophagy-based secretory pathway, facilitating their exit from the cytoplasm.

The main findings of the aforementioned studies are compiled in Table 1 (in chronological order) and summarized in Figure 1.

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FIG. 1.

Illustration of the interactions between autophagy and the inflammasome. Inflammasomes are cytoplasmic multimeric protein complexes usually formed by one sensing, one adaptor, and one recruitment domains. The assembly, and hence activation, of the NLRP3 inflammasome seems to be a two stepwise process. The first step is triggered by pathogen-associated molecular patterns (PAMPs) or proinflammatory cytokines that activate their cell surface receptors (toll-like receptors [TLRs] and specific cytokine receptors, respectively), leading to the activation and nuclear translocation of nuclear factor kappa light-chain-enhancer of activated B cells (NF-κB), which increase the transcription of NLRP3 and pro-IL-1β. The second signal consists of damage-associated molecular patterns (DAMPs) that, through still incompletely characterized mechanisms (e.g., potassium efflux, cathepsin B leakage from destabilized [phago]lysosomes or mitochondrial reactive oxygen species [mtROS], and mitochondrial DNA [mtDNA] release), lead to the assembly and activation of inflammasomes. Inflammasomes catalyze the activation of procaspase-1 (pro-Casp-1), which proteolytically activates interleukin (IL)-1β and IL-18 and induces pyroptosis in the immune system cells. These two proinflammatory events seem to be counteracted by Casp-1-mediated activation of the autophagy cascade. Upon cell stimulation with PAMPs or DAMPs, autophagy first stimulates the release of IL-1β, while inhibiting pyroptosis, thus acting as a proinflammatory pathway (1). After this first outcome, evidence suggests that autophagy limits inflammasome activation through inflammasomes or pro-IL-1β degradation by autophagosomes or by the digestion of compromised organelles that otherwise would generate DAMPs (2). MVs, microvesicles; MVBs, multivesicular bodies; SLs, secretory lysosomes.

Conclusions

It is reasonable to suggest that the role of autophagy in regulating the inflammasome may depend on time and context: in the absence of a danger signal, autophagy may act to remove pro-IL-1β and inflammasome components from the cell, thus maintaining cellular homeostasis. In the presence of such a signal, and concomitantly to inflammasome activation, autophagy may act initially as a secretory pathway to spread the inflammatory response while preventing cell death by pyroptosis. In a second phase, autophagy acts in a negative feedback loop to control inflammasome hyperactivation, thus alleviating the inflammatory response (Fig. 1). The effectiveness with which autophagy handles microorganisms, microbial products, and DAMPs rules whether the outcome is directed to suppression or exacerbation of inflammation, this being of great importance in human diseases that have strong inflammatory components. Overall, we can state that a more profound understanding of inflammasomes–autophagy mutual regulation is essential to design rational therapeutics for chronic inflammatory diseases, especially for those where autophagy and inflammasome genes have already been linked, namely Crohn's disease. So far, the acquired knowledge about inflammasome–autophagy reciprocity might only support the development of preventive approaches to ameliorate or delay the progression of an inflammatory condition, as observed in age-related diseases. Human aging is accompanied by a low-grade, chronic, systemic inflammatory state designated inflammaging, and inflammaging-associated diseases are usually correlated with NLRP3 activation or autophagy decline (Chuang et al., 2014). Therefore, one might suggest that by preserving the two processes balanced (i.e., decreasing NLRP3 activation and increasing autophagy) to maintain inflammation at physiological levels, life span could be improved. This is somehow corroborated by the fact that some natural compounds (e.g., Brazilian propolis extracts; resveratrol) suggested to be anti-inflammatory have been reported to reduce oxidative stress, induce autophagy, and decrease NLRP3 activation. In summary, defective inflammasome and autophagy activity exacerbate inflammation, which is in the origin of many (chronic) disorders. Inducing autophagy to inhibit the inflammasome and excessive inflammation or targeting directly specific NLRs to reduce their activity seems to be a promising approach to revert the inflammatory process; however, this is a delicate goal to achieve due to the cross talk between inflammasomes and autophagy, which can be easily compromised.