Inflammasome Biology: Fundamentals, Role in Disease States, and Therapeutic Opportunities

Inflammasome Biology: Fundamentals, Role in Disease States, and Therapeutic Opportunities is a complete reference on the role of inflammasomes in health and disease. Sections cover the different types of inflammasomes, including cellular signaling, structural and evolutive aspects, overview the role of inflammasomes in key diseases, microbial infections and human body systems conditions, cover the interplay between Inflammasomes and cell death processes, and discuss current therapeutic opportunities driven by inflammasome research, including targeting, blocking and inhibiting the development of inflammasomes through both synthetic and natural compounds.

This book is the perfect reference for cell biologists, immunologists and research clinicians to understand the foundations of inflammasomes and explore the therapeutic opportunities they present. Pharma researchers may also find this reference invaluable in devising new approaches to developing anti-inflammatory drugs.

• Provides comprehensive coverage of the subject of inflammasome biology
• Authored by leading experts worldwide
• Provides biological insights that have both health implications and therapeutic potential

• Language: English
• Publisher: Academic Press
• Release date: Nov 23, 2022
• ISBN: 9780323972062

Book preview

Section 1

Fundamentals of inflammasome biology

Outline

Chapter 1. The inflammatory process at the cellular level

Chapter 2. Inflammasome formation and triggers

Chapter 3. The NLRP1 and CARD8 inflammasomes

Chapter 4. Cellular signaling, molecular activation, and regulation of the NLRP3 inflammasome

Chapter 5. Cellular signaling, molecular activation, and regulation of the NLRP6 inflammasome

Chapter 6. Molecular regulation of NAIP/NLRC4 inflammasomes

Chapter 7. Cellular signaling, molecular activation, and regulation of the AIM2 inflammasome

Chapter 8. Molecular activation, cellular signaling, and regulation of the Pyrin inflammasome

Chapter 9. Cellular signaling, molecular activation, and regulation of the noncanonical inflammasome

Chapter 10. Cellular signaling, molecular activation, and regulation of auto-active inflammasomes: Insights from disease-associated variants

Chapter 11. Autophagy and the inflammasome

Chapter 12. Inflammasome effector functions: a Tale of Fire and Ice

Chapter 13. Inflammatory caspases

Chapter 14. Structural aspects of inflammasomes forming NOD-like receptors

Chapter 15. Evolutive aspects of inflammasomes

Chapter 1: The inflammatory process at the cellular level

Francesco Di Virgilio, and Anna Lisa Giuliani     Department of Medical Sciences, University of Ferrara, Ferrara, Italy

Abstract

Inflammation, the fundamental mechanism evolved in virtually all multicellular organisms to preserve internal homeostasis and well-being, is based on an intricate array of soluble messages and cellular interactions. This highly dynamic network is decoded by a wealth of membrane-expressed and cytoplasmic sensors that turn on/off a multiplicity of feed-forward/feed-back effector systems (immune and tissue parenchymal cells). Physiologically, successful inflammation leads to pathogen elimination or damage repair, and therefore to near restitutio ad integrum, but at times a pathological evolution may lead to unresolving inflammation, and thus to chronic inflammatory diseases such as autoinflammation or autoimmunity.

Keywords

Homeostasis; Immunity; Infection; Inflammation; Injury

1. Introduction

Inflammation is the fundamental response by which we react to agents, whether of endogenous or exogenous origin, endangering our body homeostasis. Inflammation is often described and dealt with as a (partially) separate process from immunity, but this is an incorrect assumption because inflammation and immunity are one and the same defensive response supported by the same cell types, largely modulated by the same soluble factors, and often ignited by the same stimuli. Leading actors in inflammation are the inflammatory (or immune) cells. As early as the 19th century, it is known that inflammatory cells migrate from blood into tissues in response to inflammatory stimuli according to an active process referred to as diapedesis (from the ancient Greek: διαπδησις leap through) (see Fig. 1.1). We now know that this apparently elementary process is the result of a highly regulated response that is at the basis of the overall mechanism of reaction to pathogens and therefore at the basis of immunity.

Inflammation has attracted attention and has been associated to human diseases since the dawn of medical practice in Ancient Egypt [1], and then in Classical Greece and in Ancient Rome, as documented by the Edwin Smith and Ebers papyruses (circa 1550 BCE) and by the works of Hippocrates (circa 460–370 BCE) and Galenus (129–circa 216 AD) that culminated in the definition of the famous four cardinal signs of inflammation (tumor, rubor, calor, dolor, the two latter indeed symptoms rather than signs) by the Roman naturalist Aulus Cornelius Celsus (circa 25 BC–50 AD) [2]. Hippocrates reported several events characterizing inflammatory lesions, among which the formation of pus at sites of wounds. The origin and the pathophysiological significance of this and other inflammation-associated events has been a focus of interest and investigation ever since.

At the birth of modern medicine there was a hot debate on the pathophysiological meaning of inflammation, and while many believed that it was a morbid phenomenon, i.e., a disease by itself, some physicians, for example, the Scottish surgeon John Hunter, modernly interpreted inflammation as a body response to an injury rather than a disease (see Ref. [3]).

It has been known for over 150 years that mammals host cells specialized to fight intruding pathogens, and that are resident in the tissues or are recruited at sites of infection or injury. In 1865, Max Schultze first described four different types of leukocytes in human blood, corresponding to those which are now known as lymphocytes, monocytes, neutrophilic and eosinophilic polymorphonuclear granulocytes, and even documented the engulfment of extracellular particles by these cells [4]. Later, Paul Ehrlich developed a staining method that allowed the unequivocal recognition of different types of leukocytes [5], and Eli Metchnikoff demonstrated the crucial role of phagocytosis in host defense (see Ref. [6]).

Figure 1.1  Drawing showing the diapedesis of inflammatory cells across the blood vessel wall at an inflammatory site. (from L. Krehl und F. Marchand "Handbuch der Allgemeine Pathologie, Hirzel Verlag, Leipzig 1924).

In the early 19th century, German pathologists were hotly debating the origin of the mucus globules or purulent globules commonly found at sites of inflammation [7]. It was a common observation of physicians and pathologists that wounds were often infiltrated by a yellowish, creamy, smelly matter referred to as pus. The process underlying the formation of pus (from the latin word pus-puris, which originated from the Greek πυον, fetid matter) was a special focus of interest in German medical science. The consolidated opinion (Virchow's opinion) held that these cells originated locally from tissue cells (epithelial or connective tissue cells) undergoing an altered differentiation (altered circumstances, altered cells) [7], although Augustus Volney Waller had already shown in 1846 that pus globules originated from white corpuscles emigrated from the blood [8]. However, we owe the unequivocal identification of pus cells as cells emigrated from blood vessels to Julius Cohnheim in 1867, a discovery highlighted by his vivid statement "ohne Gefässe, keine Entzündung, without vessels, there is no inflammation, which incidentally is the first clearcut statement highlighting the crucial importance of circulation in the pathogenesis of inflammation (the so-called angiophlogosis) [9]. This seminal contribution shifted our understanding of inflammation from a static, local," event to the dynamic process that we know and investigate today. Conheim's observations and its unorthodox interpretation (in Virchow's laboratory the belief that inflammatory cells originated locally from parenchymal cells, maybe from connective tissue, was kind of a dogma [10]) was a spectacular breakthrough and a harbinger of unprecedented developments, and of course a pillar of modern pathology and immunology. Together with the implementation of novel staining techniques introduced by Paul Ehrlich, the discovery that inflammatory cells travel across the vessel walls to reach inflammatory sites opened the way for a full understanding of the origin, differentiation, and biochemical features of inflammatory cells.

2. At the dawn of inflammation

We now know that nearly all tissues host resident inflammatory cells that are switched on by inflammatory stimuli and act in concert with blood-recruited cells to start and amplify inflammation. Incidentally, the observation that tissue-resident cells undergo morphological changes at inflammatory sites might be a reason why Rudolf Virchow concluded that inflammatory, deranged cells, originated locally, at the site of injury, from precursor cells: altered circumstances, altered cells (see Ref. [7]). Students of the History of Medicine argue that the stubborn support of the localist theory of the origin of inflammatory cells depended on Virchow's firm belief that any cell derived from a precursor cell ("omne cellula e cellula), against previous theories advocating that pathological changes (among which inflammation) were due to undefined humoral factors, such as the blastema. The blastema was an undefined medium that in the late 18th century medicine was meant to indicate the liquid environment surrounding living cells, rather than clumps of undifferentiated cells as in modern embryology (see Letourneau [11] in the translation by William Maccall). However, and rather ironically, we now know that, contrary to Virchow's derogatory belief, the liquid inflammatory blastema (that we may identify today with the biochemical inflammatory microenvironment") has a fundamental role in the process of inflammatory cell activation and differentiation.

Innate immune cells are the first elements evolved in multicellular organisms to fulfill a defensive role, while cells of adaptive immunity appeared only about 500 million years ago, at the time of the separation of agnatha (jawless fishes) from the vertebrate evolutionary tree. Thus for the most part of the existence of multicellular organisms protection against pathogens was based on native immune cells (macrophages, neutrophils, mast cells, natural killer cells, innate lymphoid cells) and on elementary recognition receptors such as the pattern recognition receptors (PRRs) [12]. Innate immune cells have kept their key function in immunity as prime effectors of chronic diseases which run in the apparent absence of a triggering antigen (e.g., the autoinflammatory diseases), as well as indispensable partners of adaptive immune cells in pathologies driven by antigens, whether exogenous or endogenous. Thus, innate immune cells participate in every stage of the inflammatory/immune response since they are both a main source and a target of most inflammatory mediators. In this view, it is a gross oversimplification, and a patent mistake, to describe the properties and functions of innate immunity without including an at least brief appraisal of adaptive immunity, and vice versa, as it is impossible to discuss inflammation at the cellular level without mentioning T and B lymphocytes, or neglecting the soluble components, whether canonical soluble mediators of inflammation (e.g., prostaglandins or kinins) or antibodies. However, an attempt can be made to provide a simplified picture identifying those basic cellular responses that ignite the overall inflammatory response and set the stage for the ensuing, integrated, and more complicated processes ending up with pathogen clearance, wound healing, and resolution.

Comprehension of the basic early events of inflammation may also allow identifying the factors leading to a distorted evolution causing unresolving inflammation, allergy, autoinflammation, or autoimmunity, with all the associated complications, irreversible fibrosis and cancer included. Therefore, a topical issue is how is inflammation started, or more plainly how inflammatory cells (tissue-resident innate immune cells, or in a subset of conditions blood mononuclear and polymorphonuclear leukocytes) integrate the different inputs delivered by pathogens (pathogen-associated molecular patterns, PAMPs) and by the injured tissues (damage-associated molecular patterns, DAMPs).

3. Detection and integration of inflammatory signals

As is well known, inflammation is one of the most important homeostatic systems that preserve body well-being against endogenous and exogenous potential insults. To perform this task, inflammation is modulated by a finely tuned network of feed-forward and feed-back signals that act upon a complex array of cellular throttle/brake mechanisms. Inflammation is a very powerful weapon, and like any potentially destructive device, should be unleashed only when absolutely needed, i.e., when tissue homeostasis is seriously altered and the overall body well-being endangered. As stated by Carl Nathan in 2002 Each cell commits to recruit and activate others based on multiple inputs, generally requiring evidence of both injury and infection [13], but how is this evidence collected and integrated with other signals to ignite (or abort) inflammation? A detailed knowledge of the events and circumstances occurring during the first phases of inflammation is of paramount importance because it is widely acknowledged that chronic inflammatory diseases (e.g., autoinflammatory or autoimmune diseases) are context-dependent, implying that changing the local physiological conditions might skew the response to a given injurious stimulus toward different outcomes. This may explain why even in the absence of known predisposing genetic factors, the same agent may trigger utterly different responses such as a physiological, self-resolving, acute inflammation, or a long-lasting chronic inflammation, or an allergic reaction, and even tolerance and immunosuppression. For example, knowledge of the local early inflammatory context might help understand why a local insult, e.g., a physical trauma like the Koebner phenomenon, may precipitate a disease such as psoriasis with features of both autoimmunity (i.e., antigen-triggered) and autoinflammation (i.e., not antigen-triggered).

Throughout our life, pro-inflammatory stimuli are constantly released into our body, whether due to occasional intrusion of extracellular microorganisms, to the constitutive close cohabitation of bacteria with body cells in the intestinal mucosa, or to accidental minor traumas. Normally, these events run fully asymptomatically since they are efficiently dealt with locally, with no substantial impact on overall systemic body homeostasis. However, under certain conditions, whether due to an excessive release of PAMPs and DAMPs, or to a mistaken interpretation of these signals by immune cells, the threshold for activation is exceeded, and full-blown inflammation is initiated. Inflammation is basically a matter of communication, i.e., of signal exchange. At the most elementary level, four components are needed: a source, a messenger, a sensor, and a target/effector [14].

Sources of inflammatory stimuli are pathogens (metazoan parasites, fungi, protozoa, bacteria, viruses) as well as stressed, injured, or dying body cells. Pathogens signal their presence via PAMPs, while tissue cells signal their distress via DAMPs or via homeostasis-altering molecular processes, HAMPs [15]. We have a fairly clear picture of PAMP-sensing by immune cells, less clear is DAMP-sensing, and even less clear HAMP-sensing. However, most PRRs will bind both PAMPs and DAMPs. As initially proposed by Charles Janeway, numerous sensors, he named PRRs [16], have evolved during the ages, to highlight the crucial importance that multicellular organisms have assigned to detection of potentially dangerous microorganisms. Different classes of plasma membrane-expressed, intra-endosomal or cytosolic PRRs fit to detect extracellular pathogens as well as intracellular parasites (e.g., Legionella pneumophila or Mycobacterium tuberculosis) have been identified [17,18]. Those expressed on the plasma membrane (but also within endosomes) are the Toll-like receptors (TLRs) and the C-type lectin receptors (CLRs), while in the cytoplasm we find retinoic acid–inducible gene-I (RIG-1)-like receptors (RLRs), nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs), melanoma differentiation-associated protein 5 (MDA5), and cyclic GMP synthase (cGAS)/stimulator of interferon genes (STING) complex [19,20].

TLRs bind several different PAMPs, ranging from lipopolysaccharide (LPS) to lipoteichoic acid, from bacterial flagellin to peptidoglycan, from zymosan to CpG DNA and single strand RNA (ssRNA). CLRs bind ligands as different as α-mannans, mannosylated fatty acids, β-glucan, and keratin or F-actin released from dying cells. RLRs and MDA5 are cytosolic viral sensors driving type I IFN-dependent responses, while cGAS/STING recognize cytoplasmic double strand DNA (dsDNA) to promote the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. NLRs bind a heterogenous mix of agents such as alum, uric acid, bacterial flagellin, amyloid-β, asbestos, silica, and Bacillus anthracis lethal toxin. Some members of the NLR family are components of the inflammasome, a key intracellular organelle integrating inflammatory signals (Fig. 1.2), and participate in its activation, while others fulfill important functions in immunity beyond inflammasome signaling. Best known inflammasomes are based on NLR scaffold (i.e., NLRP1, NLRP3, NLRP6, NLRC4), but two non-NLR inflammasomes are also known, the absent-in-melanoma-2 (AIM-2) inflammasome and the pyrin inflammasome [21]. The AIM-2 inflammasome binds dsDNA, while the pyrin inflammasome is turned on by pyrin dephosphorylation triggered by certain bacterial toxins such as toxin B from Clostridium difficile [22]. The pyrin inflammasome might be an example of a HAMPs sensor since it is turned on by a change in the intracellular environment (level of phosphorylation of a target protein) [15].

Figure 1.2  Integration of inflammatory signals: the inflammasome.

Several intracellular molecules released following injury or cell death are endowed with the ability to alarm the body (alarmins), among which high mobility group box-1 (HMGB-1) protein, extracellular cold-inducible RNA-binding protein (eCIRP), heat-shock proteins (HSPs), extracellular RNA (eRNA), cell-free DNA (cf-DNA), and extracellular ATP (eATP) are best known [23]. Cell surface receptors for DAMPs include TLRs, NLRs, receptors for advanced glycosylation end-products (RAGEs), and the P2X7 receptor (P2X7R) for eATP. Receptors for DAMPs are in general less ligand-specific compared to those for PAMPs as both TLRs and RAGEs can function as sensors for different DAMPs. A relevant exception is the P2X7R that is highly selective for eATP [24]. More recently, the triggering receptor expressed on myeloid (TREM) cells has also been suggested to be a sensor for extracellular actin and HMGB1 protein, and therefore in principle a sensor for DAMPs [25], but its role in danger sensing is as yet poorly characterized. Signals generated by the different PRRs are transmitted by diverse and often interacting intracellular pathways and converge on a rather limited set of transcription factors such as NF-κB, interferon regulatory factors (IRFs), nuclear factor of activated T cells (NFAT), and activator protein 1 (AP1) [18]. Thus, a main issue at the cellular level is how innate immune cells integrate the different inputs received by the different PRRs, which are often activated by extracellular as well as intracellular PAMPs or DAMPs. In the real life, macrophages are exposed to the combined action of extracellular PAMPs (e.g., LPS) released by infectious agents, and DAMPs released from injured tissues. Furthermore, bacterial products accumulate in the phagosome as a result of phagocytosis, bacterial killing and digestion, and may be released into the cytoplasm, often together with DAMPs, not to mention that bactericidal factors generated by pathogen-stimulated macrophages, e.g., reactive oxygen species (ROS), have profound effects on macrophage responses besides contributing to pathogen clearance.

Accumulation of eATP at inflammatory sites is also relevant for the progression toward chronic inflammation or resolution and repair. In fact eATP is the main source of extracellular adenosine, a nucleoside that on one hand has a potent vasoactive activity, thus supporting the first tissue phases of inflammation, and on the other hand has a remarkable anti-inflammatory activity inhibiting neutrophil recruitment, and M1 macrophage and T-lymphocyte activation [26].

Many PAMPs trigger secretion of preformed inflammatory mediators (for example, histamine from mast cells) or stimulate ready to go effector systems leading to the neo-synthesis and release of additional early acting mediators. Bacterial products such as formylated peptides (e.g., the small fMetLeuPhe peptide) and LPS drive NADPH oxidase activation and a large accumulation of ROS which are instrumental for bacterial clearance and at the same time modulate macrophage immune functions [27]. Some bacterial products are also by themselves potent stimulants of the release of arachidonic acid metabolites, but others require co-stimulation by additional agents providing an accessory stimulus, as for example an increase in the intracellular Ca²+ concentration [28]. Co-operation between two distinct agents (a priming factor and a triggering stimulus) in the ignition of inflammation is also well exemplified by IL-1β secretion where a triggering DAMP (eATP) is needed to achieve optimal cytokine secretion from PAMP (LPS)-primed macrophages [29]. Mutatis mutandis, the LPS/eATP cooperation epitomizes Nathan's statement that full activation of inflammation requires evidence both of the presence of a pathogen (LPS) and of tissue damage (eATP) [13]. In general, although there are cases where inflammation can be induced prospectively by PAMP-sensing in the absence of DAMP generation, in most cases inflammation is indeed triggered retrospectively, after tissue damage has occurred (see Ref. [30] for an insightful review of the essential features of inflammation). There is a pathophysiological rationale for a two-step mechanism for activation since if a pathogen is not actually dangerous (as signaled by the lack of release of DAMPs), there is no need to mount a potentially destructive response.

4. Inflammatory cells and inflammatory mediators

Tissue-resident immune innate cells such as macrophages and mast cells are the first elements activated by injury or infection. Mast cells are fast-responding cells equipped with a vast repertoire of receptors designed to ligate neurotransmitters released by stimulated or injured nerve terminals, by DAMPs and PAMPs. It is a long-known, albeit often neglected, finding that eATP is one of the most potent histamine-releasing agent from mast cells [31]. Release by mast cells of histamine, platelet-activating factor (PAF), eicosanoids, and other inflammatory mediators is a fundamental contribution to the early inflammatory microenvironment that molds reactivity of resident inflammatory cells and promotes recruitment of additional cells from circulation. In these early stages transcription of cytokine genes and of the machinery for their release is started.

Local accumulation of PAMPs and DAMPs, or generation of HAMPs, switches on the inflammasomes, either by directly binding to the leucin-rich repeat (LRR) domain of the NLR scaffold proteins, or via recruitment of plasma membrane receptors (e.g., the P2X7R in the case of the NLRP3 inflammasome), or via perturbation of the intracellular milieu (e.g., efflux of cytoplasmic K+ or pyrin dephosphorylation, which promote activation of the NLRP3 and pyrin inflammasomes, respectively) [32]. Multiple inflammasome subtypes catalyze procaspase-1 conversion into the enzymatically active caspase-1 that in turn promotes two fundamental steps at the inception of inflammation: a) maturation of pro-IL-1β into IL-1β, and b) cleavage of full length gasdermin D into the membrane-targeted N-terminal fragment. Mature IL-1β is the biologically active form that promotes expression of adhesion molecules by endothelial cells, migration and activation of inflammatory cells recruited from circulation and systemic changes associated with inflammation (e.g., fever, anorexia, cachexia). Priming of immune and nonimmune cells by IL-1β enhances the effects of later released cytokines. Oligomerization of cleaved gasdermin D fragments into the plasma membrane generates aqueous pores that may lead to pyroptosis, a caspase-1-dependent, highly inflammatory, form of programmed cell death [33]. Gasdermin pores are freely permeable to IL-1β, IL-18, and other cytosolic components, and is thus suggested that they are a main mechanism for the release of these highly inflammatory cytokines, although other nonpyroptotic mechanisms for IL-1β release have also been described. Early inflammasome activation and opening of the gasdermin pore is crucial since these events promote not only release of IL-1β but also of DAMPs that further amplify inflammation.

Cell stress or injury are causes of drastic perturbation of the intracellular milieu. Therefore it is not surprising that, as hypothesized as early as 1996 to explain the mechanism of IL-1β maturation and release, at a time when the discovery of the inflammasome was still far away [34], a decrease in cytoplasmic K+ concentration is decoded by innate immune cells as a strong proinflammatory signal, and exploited to drive inflammasome activation (at least of the NLRP3 and NLRP1 subtypes) [35]. In this sense, the NLRP3 inflammasome may be considered a sensor of imbalances in the intracellular ion homeostasis, and therefore a sensor of HAMPs [32].

Together with IL-1β other important ingredients of the inflammatory soup such as cytokines (e.g., TNF, IL-6, and IL-12) and chemokines (e.g., IL-8/CXCL8) are released from macrophages and from local professional antigen-presenting cells (APCs) during these early phases. Parenchymal, noncanonical inflammatory cells actively participate in the generation of this highly pro-inflammatory milieu. For example, keratinocytes both produce and respond to IL-1β, IL-18, TNF, IL-6, IL-12, IL-23, IL-7, IL-15, IFNs, chemokines, and growth factors [36]. Lung alveolar type II epithelial cells (type II pneumocytes) are another instructive example since these cells release IL-1β, TNF, IL-6, and chemokines, thus actively contributing to the pathogenesis of lung inflammatory diseases.

While there is no doubt that canonical inflammatory cells (i.e., macrophages, DCs, polymorphonuclear leukocytes, and mast cells) are pivotal in the ignition of inflammation and its amplification, there is also no doubt that the specific anatomical microenvironment is crucial for amplification and evolution. One of the most striking changes that attracted the attention of early physicians and pathologists was the increased cellularity at inflammatory sites (see the seminal observations of Julius Cohnheim [7] referred to earlier in this chapter). Teleologically (to the extent that we can think teleologically in science), one of the main aims of the local release of proinflammatory mediators is recruitment of additional inflammatory cells, thus increasing the weaponry for embank the injury or fight the infection, and predispose the ground for repair. Increased cellularity was interpreted from the very beginning as a positive indication of the body response to infection (or more in general to injury) and a harbinger of favorable evolution and healing (Galenus is even reported to believe the pus was very positive for wound healing, and to refer to it as bonum et laudabile, good and praiseworthy, although this is likely a late, medieval, misunderstanding of his medical doctrine) [37]. The main protagonists of pus formation are blood-derived polymorphonuclear granulocytes, the highly motile cells that are recruited at inflammatory sites during septic inflammation. Once thought to be short-living and almost exclusively associated to acute inflammation, we now know that their lifespan in circulation might be much longer, and that they might also be associated to chronic inflammation [38]. Thanks to their highly efficient bactericidal systems (NADPH oxidase-generated highly reactive oxygen metabolites, lysosomal enzymes, or unorthodox mechanisms such as the formation of extracellular traps generated by the release of their own DNA [39]), these cells are a fundamental asset of antimicrobial defense and a main stake in inflammation. Polymorphonuclear granulocytes express a P2X7R-activated NLRP3 inflammasome with an important role in anti-Streptococcus defense [40] and are a rich source of early-acting inflammatory mediators, of cytokines and chemokines.

With the appearance of adaptive immunity some 500 million years ago, the cellular and biochemical components of the early inflammatory microenvironment expanded their regulatory function to also include T and B lymphocytes and the overall network of host–pathogen interaction. Although, strictly speaking, adaptive immunity, with its highly specialized novel players and sophisticated network of soluble mediators, is not normally dealt with within cellular inflammation, it is clear that the specific biochemical and cellular components characterizing the inflammatory microenvironment set the conditions for processing exogenous (pathogen-derived) and endogenous (host-derived) antigens by APCs and determine their presentation to lymphocytes. Antigen-presenting ability of DCs is strongly enhanced by inflammatory cytokines and other mediators accumulating at inflammation foci (e.g., IL-1β, IFN-γ, TLR4 ligands, or NLRP3 inflammasome stimuli), thus increasing the immunogenicity of exogenous antigens, and under some extreme pathophysiological conditions, of endogenous antigens as well. For example, antigen cross presentation by DCs is boosted by inflammatory cytokines or by HSPs, thus highlighting the paramount role of local inflammation in the induction of organ-specific autoimmune diseases.

5. Inflammation is an energy-consuming process

Tight control of energy metabolism is a basic requirement of inflammation. Immune cells require ATP, glucose, fatty acids, lactate, glutamine, and other key substrates, and, despite at times they can do without, they also need oxygen. Proinflammatory M1 macrophages are more dependent on glycolysis, show a truncated tricarboxylic acid cycle and an accumulation of itaconate, while M2 rely more on oxidative phosphorylation [41]. Naïve and activated CD8+ T lymphocytes depend on oxidative phosphorylation and on aerobic glycolysis, respectively, and in general the preferred pathway for ATP generation may change during differentiation, adapting immune cells to each given task to be fulfilled. A thorough understanding of metabolic modulation of immune cells during inflammation will be crucial for understanding the evolution of the immune responses and prevent harmful inflammation.

6. Resolution (or lack of resolution) of inflammation

As for any homeostatic mechanism, activation of pro-inflammatory (feed-forward) pathways is closely associated to that of anti-inflammatory (feed-back) pathways. If successful, anti-inflammatory pathways will lead to progressive attenuation of the inflammatory response, tissue regeneration and healing. An obvious absolute prerequisite is removal of the triggering stimulus, often the result of the activity of the pro-inflammatory agents released by activated immune cells. For example, resolution of inflammation is accelerated by killing of microbial pathogens by ROS generated by neutrophil NADPH oxidase, or by removal of dead cells and extracellular debris by macrophages. Contextually, although with some lag, specific anti-inflammatory pathways are also activated. Genes encoding IL-1β, TNF, and other major inflammatory mediators are progressively silenced, while others encoding anti-inflammatory factors, for example, IL-1 receptor antagonist (IL-1Ra), IL-10, TGF-β, soluble IL-1 and TNF receptors are induced.

Specialized proresolving mediators (SPMs) are increasingly recognized to have a key role in the resolution of inflammation [42]. It is understood that after the initial burst of release of pro-inflammatory mediators, innate immune cells undergo a lipid mediator class switch (although increasing evidence suggest that also canonical arachidonic acid metabolites may have an anti-inflammatory activity [43]) that leads to the synthesis of poly-unsaturated fatty acid (PUFA) derivatives with anti-inflammatory activity. Specific lipoxygenases promote the synthesis of arachidonic acid metabolites named lipoxins, and of omega-3 fatty acid–derived metabolites such as resolvins, maresins, and protectins [44]. Both lipoxins and resolvins inhibit chemotaxis, cytokine release, as well as leukotriene-dependent cell stimulation. Following the full stimulation of adaptive immunity, antibody-mediated antigen removal will also greatly hasten resolution of inflammation and tissue repair.

An important role in wound repair and in the resolution of inflammation is also played by the M1/M2 macrophage phenotypic switch. M2 macrophages express the decoy IL-1 receptor and secrete the IL-1Ra, thus effectively antagonizing IL-1β, and the fibrogenic cytokine TGF-β. M2-polarized macrophages also express high levels of immunosuppressive factors such as arginase 1 (ARG1) [45]. Transition of the classical pro-inflammatory M1 macrophages to the alternatively activated M2 phenotype mitigates local inflammation and promotes angiogenesis, lymphangiogenesis, collagen deposition, and ultimately tissue repair. A powerful stimulus to resolution of inflammation is also provided by the accumulation of adenosine that promotes the M1/M2 shift, and triggers the angiogenic switch and fibrosis [46].

Lack of removal of the triggering stimulus causes a persistent stimulation of inflammatory cells, that can be further aggravated by genetic predisposition as in autoimmune or autoinflammatory diseases. Chronically activated macrophages and T lymphocytes release large amounts of fibrogenic growth factors such as PDGF, FGF, TGFβ, as well as cytokines such as IL-1β, IL-4, and IL-13, and at the same time undergo a shift in their ability to synthesize and release metalloproteinases, which lead to fibroblast activation and fibrosis [47]. Fibrosis, to a lesser or larger extent, is an unavoidable and serious consequence of chronic inflammation causing irreversible changes in tissue anatomy and function. Presence of large undigestible extracellular material may promote the formation of characteristic cellular syncytia such as the multinucleated giant cells observed in the typical inflammatory granulomas generated by foreign body response or in Mycobacterial infections [48]. Multinucleated giant cells, a rich source of cytokines such as IL-1β, TNF, and TGFβ [49], originate from the fusion of macrophages activated by inflammatory cytokines (e.g. IL-4, IFN-γ, and TNF), apparently as a consequence of an unsuccessful phagocytic effort. The NLRP3 inflammasome is involved in the early inflammatory response to foreign material, but the NLRP3 or NLRC4 proteins themselves are dispensable for further progression to formation of multinucleated giant cells [50]. On the contrary, there is a requirement for apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) and caspase-1. It is unclear why only some components of the canonical inflammasome complex are required to permit cell fusion and full generation of the final cell syncytium. AIM2 and NLRP1 are known to form ASC-containing inflammasomes, thus it is possible that additional inflammasome subtypes participate in the formation of multinucleated giant cells and of inflammatory granulomas.

7. Conclusions

Inflammation is a complex biological process with a fundamental defensive and homeostatic function that oversees the full spectrum of immune responses, from the mere detection of foreign antigens or danger signals to the activation of highly specific effector systems such as clonal T and B expansion and antibody synthesis and secretion. Full blown inflammation is based on a complex interaction of feed-forward/feed-back signals that may lead to resolution and repair or to a chronic, unremitting, response (Fig. 1.3). Furthermore, inflammation is intimately intertwined with additional, only apparently independent, homeostatic systems such as thermal and hydro mineral homeostasis: inflammation always affects the organism as a whole, or in other words inflammation is a holistic response. For this reason, an accurate knowledge of the fundamental cellular events of inflammation is the basis for a thorough understanding of the most complex systemic disease processes.

Acknowledgments

Work supported by grants from the Italian Association for Cancer Research (n. IG 13025 and IG 18581), Cure Alzheimer's Fund (USA), The Ministry of Education of Italy (PRIN n. 20178YTNWC), the COST Action 21130, and institutional funds from the University of Ferrara.

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Figure 1.3  Basics of inflammation. From left to right: (A) initiation of inflammation due to release of neuromediators, infection or tissue injury. (B) Decoding of pro-inflammatory signals by immune cells, cytokine release and inflammatory cell recruitment at inflammatory sites. (C) Inflammasome activation, further release of inflammatory cytokines, and amplification of inflammation. (D) Stimulation of release of pro-resolving mediators and resolution of inflammation.

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Chapter 2: Inflammasome formation and triggers

Iva Hafner-Bratkovič ¹ , ²       ¹ Department of Synthetic Biology and Immunology, National Institute of Chemistry, Ljubljana, Slovenia      ² EN-FIST Centre of Excellence, Ljubljana, Slovenia

Abstract

Inflammasomes are intracellular sensors of pathogens and guardians of cell well-being. Microbial, endogenous, and synthetic triggers either directly or indirectly induce the assembly of an inflammasome that consists of a receptor/sensor, an adaptor, and a proinflammatory caspase such as caspase-1. Activated caspase-1 exerts its effector functions by cleaving proinflammatory cytokines pro-IL-1β and pro-IL-18. Inflammatory caspases also cleave gasdermin D which enables its N-terminal domain to form pores in the plasma membrane. Excessive pore formation leads to ninjurin-1-mediated plasma membrane rupture and cell death called pyroptosis. Inflammasome sensors undergo quite distinct modes of activation. Some sensors such as AIM2 recognize their activators directly, others such as Pyrin sense pathogen activity instead. In this chapter, the basic principles of inflammasomes will be presented and mechanisms of activation of several inflammasomes will be explained to introduce the reader to the fascinating world of these intracellular inflammatory devices.

Keywords

Canonical inflammasome; Inflammasome; Inflammasome sensor; Noncanonical inflammasome; Proinflammatory caspases; Triggers

1. The inflammasome platform assembles in response to various triggers

20 years ago, Martinon, Burns, and Tschopp described the inflammasome, an intracellular multimolecular platform for activation of inflammatory caspases [1]. Since then, diverse inflammasomes have been discovered and shown to play an important role in the defense against pathogens and/or to drive inflammation in sterile diseases. In principle, inflammasomes consist of a sensor, an adaptor, and an effector component. Inflammasome sensors predominantly come from nucleotide-binding domain leucine-rich repeat-containing receptor family (NLR) or absent in melanoma 2 (AIM2)-like receptor family (ARL). Inflammasome assembly is initiated when inflammasome sensor responds to pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). Some inflammasome sensors are detecting perturbations in cell homeostasis—homeostasis-altering molecular processes (HAMPs) [2]. Direct and indirect triggers induce posttranslational modifications, domain rearrangements, and oligomerization of the sensor protein which in turn assemble a seed for adaptor ASC (apoptosis-associated speck-like protein containing a CARD) polymerization. ASC is a bipartite protein where the pyrin domain (PYD) enables interaction with sensors and polymerization into filaments and caspase activation and recruitment domain (CARD) enables filament cross-linking and recruitment of procaspase-1 molecules [3]. After self-activation caspase-1 cleaves various substrates including proinflammatory cytokines interleukin (IL)-1β and IL-18. Another substrate of caspase-1 is gasdermin D. Cleavage of gasdermin D leads to liberation of its N-terminal domain [4,5] to form pores in the plasma membranes [6–9] thus causing pyroptosis. In this chapter, particular inflammasomes and their triggers will be briefly presented to give a broad overview of our current understanding of how triggers induce particular inflammasome assembly. Each inflammasome and distinct stages of inflammasome assembly are more comprehensively presented in the following chapters.

2. Inflammasome sensors predominantly belong to NLR and ALR receptor families

Members of the cytosolic NLR receptor family are characterized by central nucleotide-binding and oligomerization domain (NBD) and C-terminal leucine-rich repeat domain (LRR). NLRs are members of signal transduction ATPases with numerous domains. NBD domain together with associated domains harbors conserved motifs that enable nucleotide binding. Together these domains are called NACHT domain (present in NAIP, CIITA, HET-E, and TP1) [10]. The NACHT domain acts as an oligomerization domain and ATPase activity is important for NLRP3 signaling [11,12]. The leucine-rich repeat domain (LRR) acts as ligand recognition and binding domain in Toll-like receptors and also in NLRC2 (NOD2), whereas in the case of NLRC4 it seems to have regulatory function disabling activation in the absence of the triggers [13]. Crucial for NLR function as an inflammasome sensor is the effector domain. Based on evolutionary analyses and which effector domain is present [14], NLR proteins are divided into several groups, such as NLRP family containing a pyrin (PYD) domain or NLRC family containing a CARD domain. PYD and CARD domains belong to the death fold domains, which are characterized by six tightly packed α-helices in a Greek-key fold. Death fold domains are interacting with other death fold domains via homotypic or heterotypic interactions. NAIPs (NBD-domain-containing inhibitor of apoptosis proteins) are NLR proteins that are characterized by the N-terminal baculoviral inhibitor of apoptosis protein repeat (BIR) domains. BIR domains do not belong to the death fold, thus NAIP proteins engage another member of NLR family, NLRC4, for inflammasome assembly. Versatile mammalian NLRs exist, however not all function as inflammasome sensors. Inflammasome-forming sensors NLRP1, NLRP3, NLRP6, and NAIP/NLRC4 will be presented later in more detail. Some other sensors were already shown to form inflammasomes such as NLRP9. In addition, inflammasome-independent functions are emerging for known inflammasome sensors and other NLRs [15].

ALRs appeared relatively late during evolution and are present in placental and marsupial mammals, with the human genome encoding 4 members of this family and the mouse genome 13 members [16]. ALRs or PYHINs are characterized by two main domains, a HIN domain and a PYD domain. The HIN domain is composed of two tandem oligonucleotide/oligosaccharide binding (OB)-folds [17]. OB-fold is 70–80 amino acid β-barrel of five or six strands. HIN domain of ALR has been shown to bind DNA [18]. AIM2 via its HIN domain detects DNA in the cytosol. In contrast to NLR proteins, ALRs do not harbor domains capable of oligomerization, however, DNA acts as an oligomerization platform for AIM2, releasing autoinhibitory interactions between HIN and PYD domains. ASC is then recruited via homotypic PYD–PYD interactions and inflammasome is assembled as described above [19–22]. In contrast to cytosolic AIM2, other human ARLs such as IFI16 (IFN-γ inducible protein 16) are predominantly present in the nucleus [19], yet have emerging functions in innate immunity.

3. Adaptor ASC augments inflammation

Upon trigger sensing various mechanisms described later lead to receptor or its effector domain arrangement that in the case of PYD-containing receptors makes a proper nucleus for adaptor ASC recruitment and polymerization. Upon binding to a receptor, ASCPYD domains form filaments that are not amyloid-like and ASC α-helical secondary structure is retained [23]. ASCPYD filament formation is important for inflammasome signaling as mutations that disrupt filament formation do not support inflammasome activity [23]. ASCCARD domains connect ASCPYD filaments together into ASC speck, a micrometer-sized fur ball-like complex in the perinuclear region. ASCCARD domain is also crucial for the recruitment of procaspase-1 molecules downstream of PYD-containing receptors [3]. Upon pyroptosis, ASC specks can be released from the cells and serve as caspase-1 activating complexes in the extracellular space [24,25]. Furthermore, ASC specks can reenter phagocytes and either cause lysosome destabilization and NLRP3 inflammasome activation or upon reentering the cytoplasm recruit naïve ASC monomers and thus perpetuate inflammation in the absence of sensor oligomerization [24,25]. Extracellular specks were found in autoinflammatory conditions such as CAPS [25], chronic obstructive pulmonary disease, and pneumonia [24]. Interestingly, ASC-specific antibodies from mice with experimental lupus did not neutralize ASC speck-mediated inflammation but rather perpetuated it via enhanced Fc receptor-mediated phagocytosis of ASC specks. Nanobodies were on the other hand able to disaggregate extracellular ASC specks and ameliorate inflammation in arthritis mouse models [26]. Further studies will need to address how significant the participation of extracellular ASC specks is in supporting inflammation in various diseases.

CARD-containing receptors could in principle signal without ASC by binding and activating procaspase-1 molecules directly. However, only CARD8 was shown to signal without ASC [27]. In spite of a similar activation mechanism (described later), NLRP1 was shown to depend on ASC for activation [27]. ASC specks also appear after NAIP/NLRC4 inflammasome formation and enhance cytokine maturation while in this case, pyroptosis does not depend on ASC speck formation [3].

4. Caspase-1 processes proinflammatory cytokines and gasdermin D

Caspase-1 upon activation drives at least two proinflammatory responses. Its known substrates are proinflammatory cytokines pro-IL-1β and pro-IL-18. Cleavage of pro-IL-1β and pro-IL-18 yields active forms of cytokines that orchestrate defense responses and modulate adaptive immunity [28]. Mature IL-1β is released from the cells by various mechanisms. The binding of IL-1β to IL-1R1 enables the receptor to bind the coreceptor IL-1R3. The formation of the trimeric complex enables recruitment of Myd88 and engagement of the downstream pathways, similar to TLR signaling. The major outcomes of IL-1β signaling are the production of cytokines, chemokines, and nitric oxide, expression of adhesion molecules, and cyclooxygenase type 2 (COX2) induction [28]. While expression of pro-IL-1β is induced by priming, pro-IL-18 is constitutively expressed in various cells and tissues. IL-18 molecule binds to receptor IL-1R5 that forms a complex with coreceptor IL-1R7. As in the case of IL-1β signaling pathways, Myd88 is driving downstream signaling. Despite the engagement of similar signaling pathways, IL-18 does not induce COX2 and fever but instigates IFN-γ secretion from natural killer cells and T lymphocytes [28].

The second pathway as the consequence of inflammasome and caspase-1 activation is necrotic cell death called pyroptosis. The mechanism of inflammasome-triggered cell death was unknown for a long time until in 2015 two studies led by Vishva Dixit [5] and Feng Shao [4] discovered that gasdermin D was the substrate of inflammasome-triggered caspases, necessary for pyroptosis. In the absence of protease activity, full-length gasdermin D consists of an N-terminal domain and a C-terminal domain that are connected with a linker. Caspases-1 and -11 cleave after residue 276 and some other proteases cleave at other positions within the linker region. Within full-length gasdermin D, the C-terminal domain interacts with the pore-forming N-terminal domain thus preventing pore formation [6–9]. The N-terminal gasdermin D (1–276) binds to the negatively charged lipids such as phosphoinositides and cardiolipin [6,7], inserts into the membrane, and forms pores that are able to release molecules smaller than the inner diameter of the pore [29,30]. Additionally, the negatively charged interior of the pore permits faster release of positively charged and neutral cargoes [29]. Besides proteolytic cleavage, gasdermin D is also regulated at the transcriptional level via IRF2 [31] and by other posttranslational modifications such as succination [32]. Two genome-wide screens identified that gasdermin D activity is also regulated by Ragulator/Rag complex that is otherwise known to regulate metabolism in response to nutrient availability [33,34]. Additionally, ESCRT-III membrane repair can pinch off already formed gasdermin D pores [35].

Although excessive plasma membrane poration will lead to ballooning of the cell, the membrane will not crack if the protein ninjurin-1 is not expressed. Ninjurin-1 leads to plasma membrane rupture after the cell is already severely damaged by gasdermin pores [36]. Ninjurin-1 is ubiquitously expressed two-pass transmembrane protein with both termini exposed on the surface of the cell. Kayagaki et al. [36] showed that overexpression of ninjurin-1 is able to kill HEK293 cells and identified a segment at the N-terminal part to be responsible for inducing plasma membrane rupture. This segment is predicted to form an amphipathic α-helix that could disrupt the membrane in a way similar to antimicrobial peptides.

So far, I have introduced inflammasomes and inflammasome activation steps that are common to all canonical inflammasomes. Inflammasome assembly is schematically presented in Fig. 2.1. The rest of this chapter will be devoted to particular inflammasome activation, focusing on the triggers and current understanding of the mechanism of activation. Specific inflammasomes will be presented in more detail in the following chapters.

5. NLRP3 inflammasome

This fascinating molecule represents a prototypic NLR, with a pyrin domain at its N-terminus. NLRP3 was shown to be regulated at transcriptional, posttranscriptional, and posttranslational levels [37–40 ]. As is true for some other inflammasomes, two signals are needed for its activation. The first signal (priming) induces the expression of NLRP3 and pro-IL-1β [38] and via posttranslational modifications of NLRP3 [39] prepares the system for further activation with the second signal. Different posttranslational modifications particularly ubiquitination and phosphorylation at different sites were shown to regulate NLRP3 inflammasome (recently reviewed in Ref. [40]). The second signal is provided by a versatile company of triggers that include ATP, pore-forming toxins, aggregates, crystals, nanoparticles, microbes, and their components and some drugs such as imiquimod. Taking the versatility of NLRP3 triggers into account, it is highly unlikely that those DAMPs and PAMPs are actually inducing inflammasome assembly by direct binding to NLRP3. Instead, the decrease of intracellular K+ was shown to be downstream of most activators [41], although K+ efflux-independent triggers also exist [42]. Many physiological processes were shown to support NLRP3 inflammasome activation. NLRP3 was proposed to sense organelle dysfunction [43]. Particulate triggers cause lysosome destabilization [44] and plasma membrane damage [45]. Some of the activators cause damage to the mitochondria, which are a source of various NLRP3 activators and could serve as a docking platform for NLRP3 inflammasome assembly similarly to endoplasmic reticulum and Golgi apparatus (reviewed recently in Ref. [46]). Disassembly of trans-Golgi network is another process downstream of various NLRP3 triggers [47]. NLRP3 was observed to be trafficked via microtubules to microtubule organizing center where it can interact with NEK7 [48]. NEK7 was identified several years ago to be crucial for canonical NLRP3 inflammasome activation [49–51 ], and binds to multiple domains of NLRP3, where its interactions with the LRR domain might be particularly important. The complex formation forces NLRP3 into a semiactivated state and likely additional domain rearrangement is needed for NLRP3 to form the seed via PYD domains to support ASC recruitment [52]. While the LRR domain is not essential [53,54], the linker FISNA region between the N-terminal PYD domain and the NACHT domain was shown to be responsible for sensing triggers [55]. A recent study by Andreeva et al. [56] showed that NLRP3 forms inactive oligomeric double-ringed cages by LRR domains at trans-Golgi network. PYD domains are captured inside the cage and significant structural rearrangements are necessary for NLRP3 molecules to be able to interact with NEK7 and form the inflammasome. Two other studies confirmed that NLRP3 molecules form inactive oligomeric cages through LRR domain interactions