Inflammasome activation and function in liver disease.

REVIEWS Inflammasome activation and function in liver disease Gyongyi Szabo and Jan Petrasek Abstract | Inflammation contributes to the pathogenesis of most acute and chronic liver diseases. Inflammasomes are multiprotein complexes that can sense danger signals from damaged cells and pathogens and assemble to mediate caspase‑1 activation, which proteolytically activates the cytokines IL‑1β and IL‑18. In contrast to other inflammatory responses, inflammasome activation uniquely requires two signals to induce inflammation, therefore setting an increased threshold. IL‑1β, generated upon caspase‑1 activation, provides positive feed-forward stimulation for inflammatory cytokines, thereby amplifying inflammation. Inflammasome activation has been studied in different human and experimental liver diseases and has been identified as a major contributor to hepatocyte damage, immune cell activation and amplification of liver inflammation. In this Review, we discuss the different types of inflammasomes, their activation and biological functions in the context of liver injury and disease progression. Specifically, we focus on the triggers of inflammasome activation in alcoholic steatohepatitis and NASH, chronic HCV infection, ischaemia–reperfusion injury and paracetamol-induced liver injury. The application and translation of these discoveries into therapies promises novel approaches in the treatment of inflammation in liver disease. Szabo, G. & Petrasek, J. Nat. Rev. Gastroenterol. Hepatol. advance online publication 9 June 2015; doi:10.1038/nrgastro.2015.94

Introduction

Department of Medicine, University of Massachusetts Medical School, LRB 215, 364 Plantation Street, Worcester, MA 01605, USA (G.S., J.P.).

Inflammation is triggered by microbial pathogens or danger signals derived from the host. The concept of innate immunity was first proposed by Charles Janeway, who suggested that antigen-presenting cells express receptors that can sense microbial products, and that activation of these receptors initiates an inflammatory response.1 Subsequently, the ‘danger hypothesis’ was developed, in which antigen-presenting cells sense endogenous signals released by damaged cells to trigger an immune response under sterile conditions (that is, in the absence of microbial pathogens).2 These two concepts are complementary,3 and increasing amounts of evidence suggest they apply to many human conditions, including liver diseases. Many different microbial and viral signals activate innate immunity in liver disease. Hepatitis viruses are a common trigger of liver inflammation, whereas gutderived bacterial components contribute to the progression of most liver diseases. 4,5 Molecules derived from those microbial or viral pathogens are commonly known as pathogen-associated molecular patterns (PAMPs). After their translocation to the liver via portal blood, PAMPs bind to pattern-recognition receptors on immune cells in the liver, including Toll-like receptors (TLR), and initiate an inflammatory response.6–8 In addition to microbial or viral signals, liver immune cells are exposed to sterile stimuli derived from the host, which are released from damaged parenchymal and

Correspondence to: G.S. gyongyi.szabo@ umassmed.edu

Competing interests The authors declare no competing interests.

nonparenchymal cells (known as damage-associated molecular patterns, DAMPs).9 These DAMPs represent endogenous signals that, under normal circumstances, remain hidden from the extracellular environment, are released when tissues are injured and activate immune cell receptors.9 Of over a dozen identified endogenous DAMPs to date, ATP, uric acid, cholesterol crystals, amyloid beta, calcium pyrophosphate dehydrate crystals and cytosolic DNA10,11 lead to the assembly of a cytosolic protein complex termed ‘the inflammasome’, which activates the serine protease caspase‑1 (CASP‑1) and leads to the secretion of cytokines, such as IL‑1β and IL‑18.12–14 DAMPs can also induce inflammation in an inflammasome-independent manner; however, in this Review we focus on DAMP-mediated liver inflammation via inflammasome-dependent pathways. The role of the different inflammasomes in alcoholic steatohepatitis and NASH, chronic HCV infection, ischaemia–reperfusion injury and paracetamol-induced liver injury will be evaluated, as well as the ability of inflammasomes to finetune liver inflammation. Finally, we also examine the potential of inflammasomes as novel therapeutic targets in liver diseases.

Inflammasome activation

Inflammasomes are multiprotein complexes that sense danger signals via nucleotide-binding oligomerization domain receptors (commonly known as NOD-like receptors, NLRs; Figure 1). 15 NLRs contain a ligand recognition domain, a central NACHT nucleotidebinding domain (NAIP [neuronal apoptosis inhibitor

NATURE REVIEWS | GASTROENTEROLOGY & HEPATOLOGY © 2015 Macmillan Publishers Limited. All rights reserved

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REVIEWS Key points ■■ Inflammasomes are multiprotein complexes that assemble upon the sensing of danger signals and initiate the release of proinflammatory cytokines IL‑1β and IL‑18 via caspase‑1 activation ■■ By responding to low-threshold signals, the inflammasome is able to fine-tune the inflammatory response ■■ The balance between a healthy inflammatory response and chronic damage is delicate and several inflammasomes, such as NLRP3, NLRP6 and AIM2, also have a role in liver diseases ■■ Among other diseases, inflammasome activation contributes to alcoholic steatohepatitis and NASH, chronic HCV infection, ischaemia–reperfusion injury, paracetamol-induced liver injury and liver fibrosis

Signal 1

Signal 2

PAMPs

DAMPs

Crystals (uric acid, cholesterol, amyloid)

ATP K

+

P2X7 TLRs

Viral RNA Host DNA

ROS

Phagosome lysosome

Pannexin-1

Unknown ligand

Endosome Lysosome damage Cathepsin B NLRP3 ASC

ROS AIM2 ASC

TXNIP TXNIP

Pro-caspase 1

Thioredoxin

NLRP6 ASC Pro-caspase 1

Pro-caspase 1

Thioredoxin Caspase 1

NFκB

Pro-IL-1β

IL-1β

Pro-IL-18

IL-18

Nucleus

Figure 1 | Inflammasomes involvedNature in liverReviews diseases. NLRP3 and AIM2& Hepatology | Gastroenterology inflammasomes are activated in liver immune cells, the NLRP6 inflammasome is activated in intestinal epithelial cells but not in the liver. Activation of the NLRP3 inflammasome is a two-step process. In the first step, a PAMP binds to its cognate TLR, which upregulates NLRP3 and increases expression of pro-IL-1B and pro-IL‑18. A second signal is required to activate caspase‑1, which converts the precursors into mature IL‑1β and IL‑18. NLRP3 activation occurs via three separate processes: binding of ATP to P2RX7, which opens a cation channel and a large pore through pannexin 1, leading to ionic fluxes and intracellular K+ depletion; endocytosis of sterile particulates results in lysosomal damage and membrane destabilization, which activates cathepsin B and decreases intracellular pH; ROS, generated during cellular stress or cell death, initiate the detachment of TXNIP from thioredoxin and enable it to activate NLRP3. In liver disease, the AIM2 inflammasome is thought to be activated by host DNA from dying hepatocytes or viral RNA. The NLRP6 inflammasome orchestrates the colonic host–microbial interface by regulating goblet cell mucus secretion. It exerts its function predominantly via IL‑18. The ligand for NLRP6 is not known. Abbreviations: AIM2, absent in melanoma protein (also known as interferon-inducible protein AIM2); ASC, apoptosis-associated speck-like protein containing a CARD; DAMP, damage-associated molecular pattern; LPS, lipopolysaccharide; NFκB, nuclear factor kappa B; P2X7, P2X purinoceptor 7; PAMP, pathogen-associated molecular pattern; ROS, reactive oxygen species; TLR, Toll-like receptor; TXNIP, thioredoxin-interacting protein.

protein], C2TA [MHC class 2 transcription activator], HET‑E [incompatibility locus protein from Podospora anserina] and TP1 [telomerase-associated protein]) that is responsible for oligomerization, and an N‑terminal

transactivation domain.16 After its activation by inflammatory signals, NLR forms a complex with the effector molecule, pro-CASP‑1, and, in some instances, also relies on the contribution of an adaptor molecule, such as ap optosis-associated speck-like protein containing a CARD (ASC, also known as PYCARD; Figure 1). The inflammasome can then oligomerize and activates CASP‑1, which, in turn, initiates the maturation of proinflammatory cytokines IL‑1β and IL‑18.15 In addition, inflammasome activation induces chemokine expression, for example C-C motif chemokine 2 (CCL2, also known as MCP-1), which is crucial in the recruitment of bonemarrow-derived inflammatory cells to the damaged liver.17–19 Among the best-characterized inflammasomeactivating signals in liver diseases are ATP, uric acid, palmitic acid, cholesterol crystals and reactive oxygen species (ROS).20–25

IL‑1β-mediated effects Historically, the main role of inflammasomes in liver diseases has been attributed to their presence in liver immune cells and their ability to trigger inflammation via the inflammatory cytokine IL‑1β.18,26 The proinflammatory effect of IL‑1β is probably due to its synergistic action with TLR signalling that markedly amplifies inflammation via lipopolysaccharide-inducible inflammatory cytokines. 18,19,27–29 IL‑1β signalling is further strengthened by autocrine or paracrine mechanisms. For example, the inflammasome-mediated release of IL‑1β increases transcription of its own precursor (pro-IL‑1β), other inflammasome components, inflammatory cytokines and chemokines, including TNF and CCL2.17,28,30 In liver disease, IL‑1β promotes the recruitment of inflammatory cells to the liver and activates hepatic stellate cells (HSCs), which contributes to fibrosis.19,31 In addition, IL‑1β can induce triglyceride accumulation in hepatocytes18,19 and, in conjunction with TNF, triggers hepatocyte death.19,27,32 IL‑1 signalling is regulated by the IL‑1 receptor antagonist protein (IL-1RA), which is an endogenous antagonist of the pro inflammatory IL‑1β.33 IL‑1RA competes with both IL‑1α and IL‑1β at the receptor level to block IL‑1 receptor (IL‑1R1) signalling.30 IL‑1β is activated by CASP‑1 (Figure 1). This premise is supported by experiments using Casp‑1-deficient mice that lack Il‑1β activation and has been reviewed extensively elsewhere.9,34,35 However, in some studies on the innate immune response to certain bacterial infections, CASP‑1 deficiency did not prevent activation of IL‑1β. This discrepancy has been explained by the presence of other IL‑1β proteolytic mechanisms, including neutrophil elastase, proteinase‑3, cathepsin G36–41 and CASP‑11.42 However, to date, no evidence has been presented that supports the causal involvement of neutrophil elastase, proteinase‑3 or cathepsin G in IL‑1β maturation in liver diseases. IL‑18 and IL‑33 in liver diseases The inflammasome also processes substrates other than IL‑1β that might have an effect on the development of

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REVIEWS NLRP3 inflammasome

NLRP6 inflammasome

AIM2 inflammasome

LRR domains NAD domains

NLRP3

NLRP6

NACHT domains

AIM2

PYD domains CARD domains Caspase domains

HIN-200 domains

ASC

Pro-caspase-1

Figure 2 | Domain organization of inflammasome proteins. The identified Nature Reviews | Gastroenterology & core Hepatology components belong to two families, the NLR and the PYHIN family. The NLR family members relevant in the pathogenesis of liver diseases include NLRP3 and NLRP6. The PYHIN family member AIM2 is characterized by a HIN200 domain, which is involved in ligand binding, and a PYD domain. Abbreviations: AIM2, absent in melanoma protein (also known as interferon-inducible protein AIM2); ASC, apoptosis-associated speck-like protein containing a CARD; CARD, Caspase recruitment domain; HIN200, haematopoietic interferon-inducible nuclear protein with a 200 amino acid motif; LRR, leucine-rich repeat; NACHT, nucleotide-binding and oligomerization domain; NAD, NACHT-associated domain; NBD, nucleotide-binding domain; NLR, NOD-like receptor; NLRP, NACHT, LRR and PYD domains-containing protein; PYD, pyrin domain; PYHIN, pyrin and HIN200 domain-containing protein.

liver disease, such as the inflammatory cytokines IL‑18 (Figure 1) and IL‑33.43 Unlike IL‑1β signalling, IL‑18 seems to be involved in modulating the gut microbiota. In a study using a methionine-choline-deficient (MCD) diet-induced NASH model, mice deficient in Il‑1β signalling had no changes in disease severity compared with wild-type mice,44 whereas an exacerbation of NASH severity was observed in Il‑18-deficient mice on the MCD diet.44 Moreover, Il‑18 deficiency altered the gut bacterial composition in favour of a colitis-inducing microbiota, which resulted in increased translocation of bacterial products from the gut to the liver, leading to inflammation.44 The role of Il‑18 has also been studied in a mouse model of liver injury induced by the commonly used painkiller paracetamol (commonly known as acetaminophen in the USA and Japan). Il‑18-deficient mice exposed to a lethal dose of paracetamol (500 mg/kg) survived longer than wild-type controls and the survival benefit was statistically significant.45 Furthermore, in an in vitro study, monocytes could sense HCV-infected hepatocytes and responded by producing IL‑18 in an inflammasome-dependent manner.46 In liver diseases, the function of IL‑33 is largely unknown and in the few studies available to date, IL‑33 has been studied in the liver as an ‘alarmin’ signal after its release from necrotic cells.47 IL‑33 is predominantly expressed by epithelial cells, including hepatocytes,47 and activates a variety of cells: haematopoietic cells, mast cells, eosinophils, basophils, natural killer cells, natural killer T cells, CD8+ T cells, type 2 T helper (TH2) cells and nonhaematopoietic cells.48 IL‑33 operates in the nucleus and extracellular space, and appears in two forms: fulllength IL‑33 (pro-IL‑33) and mature IL‑33. The nuclear

space is the exclusive domain of intracellular pro-IL‑33, but the nuclear function of pro-IL‑33 is unclear.49 When released from the cells, pro-IL‑33 is often digested into mature IL‑33, which is not capable of localizing into the nucleus. The processing and release of pro-IL‑33 seems to be cell-type specific and several proteases, including CASP‑1, can process pro-IL‑33 into mature IL‑33.50,51 Extracellular pro-IL‑33 and IL‑33 bind to their receptor ST2, activating the myeloid-differentiation factor 88 (MyD88)-signalling pathway, which induces various cytokines and chemokines or causes cell differentiation and activation.48 Both pro-IL‑33 and mature IL‑33 promote inflammation but exhibit differences in their specific activities. Pro-IL‑33 promotes inflammation differently from mature IL‑33 in an ST2-independent fashion, whereas mature IL‑33 induces a strong TH2skewed inflammatory response. The mechanisms behind this effect are not known.52 In mouse models of alcoholic liver disease (ALD), liver fibrosis and ischaemia–reperfusion injury, Il‑33 levels were considerably increased.18,53,54 Inhibition of Il‑33 signalling decreased ischaemia–reperfusion injury by blocking activation of Kupffer cells (the resident macro phages of the liver) and NFκB,55 but accelerated carbon tetrachloride-induced liver fibrosis in another study.56 However, no difference was observed in concanavalinA‑induced acute hepatic injury between wild-type and Il‑33-deficient mice.57 Consequently, the role of IL‑33 in ALD, NASH, paracetamol-induced liver injury or viral hepatitis C is yet to be elucidated.

The NLRP3 inflammasome The NLRP3 inflammasome has been extensively studied in liver disease,9,13,58 although other types of inflammasomes have also been implicated in liver disorders, such as the absent in melanoma protein AIM2 (also known as interferon-inducible protein AIM2)19,59 and the NLRP6 inflammasome.44 In the NLRP3 inflammasome (Figure 2), the NLR component is represented by NLRP3 (NACHT, LRR and PYD domains-containing protein 3, commonly known as cryopyrin), which functions as a ligand sensor.34 When the NLRP3 inflammasome forms a complex with the adaptor molecule ASC, CASP‑1 is activated.15 Inflammasome activation is a two-step process in which a bacterial signal, for instance lipopolysaccharide, upregulates NLRP3 expression via NFκB-dependent pathways (Figure 1).60 This event enables a second signal, usually a DAMP derived from damaged cells, to activate the NLRP3 inflammasome, via one of three main pathways (Figure 1). In one of these pathways extracellular ATP signals via the P2X purinoceptor 7 (P2X7), leading to potassium efflux, recruitment of the membrane pore protein pannexin 1 and NLRP3 activation.61–63 Another mechanism involves the endocytosis of crystalized cholesterol, uric acid or amyloid. 21,61,64,65 Phagocytosis of these crystals by macrophages leads to lysosomal disruption which releases their components, including the lysosomal protease cathepsin B, which activates NLRP3 and alters intracellular pH.63 In the third mechanism,



REVIEWS Liver immune cells DAMPs PAMPs PRR

IL-1β IL-18 CCL2 TNF TGF-β1

NLRP3 AIM2 NFκB

Liver inflammation and fibrosis Hepatic steatosis; hepatocyte death

Hepatic stellate cells Liver damage Text Hepatocyte death

Liver inflammation and fibrosis

DAMPs

Hepatocytes DAMPs PAMPs PRR

IL-1β IL-18

Liver inflammation and fibrosis; pyroptosis (NASH)

Hepatic steatosis

Hepatocyte death

Cholesterol Carbohydrates

Paracetamol Ethanol

Altered gut microbiome

NLRP3

PAMPs

Gut–liver cross talk

DAMPs PAMPs PRR

TGF-β1 IL-1β IL-18

NLRP3

Intestinal epithelial cells DAMPs PAMPs PRR

IL-1β IL-18

NLRP6

Figure 3 | The role of inflammasomes in liver diseases. Absorption of high concentrations of paracetamol and ethanol can Nature Reviews | Gastroenterology result in hepatocyte death. Large amounts of carbohydrates or cholesterol can trigger hepatocyte steatosis. All&ofHepatology which can alter the composition of the gut microbiota, which results in increased microbial translocation to portal blood and elevated liver exposure to PAMPs. These gut-derived PAMPs activate liver immune cells via PRRs, resulting in increased expression of pro-IL-1b and pro-IL‑18 via a NFkB-dependent mechanism. Inflammasome activation via DAMPs converts these precursors into functional cytokines. Mature IL‑1β increases inflammatory signalling in immune cells, resulting in CCL2 and TNF upregulation. CCL2 aggravates hepatocyte steatosis and IL‑1β sensitizes hepatocytes to cellular toxicity induced by TNF. IL‑1β also activates hepatic stellate cells that further exacerbate inflammation and are a key contributor to liver fibrosis. In NASH, fatty acids activate the NLRP3 inflammasome in hepatocytes, contributing to hepatocyte death via pyroptosis. Dying hepatocytes release more DAMPs, which activate inflammasomes in immune cells, hepatic stellate cells and hepatocytes. Together, these events result in liver inflammation, fibrosis and ongoing liver damage. Abbreviations: AIM2, absent in melanoma protein (also known as interferon-inducible protein AIM2); CCL2, C-C motif chemokine 2; DAMP, damage-associated molecular pattern; NFκB, nuclear factor kappa B; NLRP, NACHT, LRR and PYD domains-containing protein; PAMP, pathogen-associated molecular pattern; PRR, pattern-recognition receptor; TGF, transforming growth factor.

thioredoxin-interacting protein detaches from thioredoxin in a ROS-dependent manner and binds to NLRP3 to trigger its activation.66,67 Whether this mechanism is a distinctly independent mode of NLRP3 activation or part of a downstream signalling process initiated by ATP or crystals, is unclear.

The AIM2 inflammasome AIM2 is a cytosolic double-strand DNA sensor, that is stimulated by bacterial, viral and mammalian host DNA to elicit CASP‑1 activation.68–70 AIM2 belongs to the family of PYHIN (pyrin and HIN domain-containing protein) proteins and contains a HIN200 domain that binds to DNA, and a pyrin domain that associates with the adaptor molecule ASC to activate CASP‑1 (Figure 2).69 AIM2 binds directly to its ligand69 and might contribute to the pathogenesis of autoimmune diseases via the recognition of mammalian DNA.71 The AIM2 inflammasome can be activated by double-stranded RNA via the helicase receptor retinoic acid-inducible gene 1, following association with the inflammasome adaptor molecule ASC, in turn leading to CASP‑1 activation.72 AIM2 has also been implicated in the response to host DNA in NASH59 and macrophage activation in ascitic fluid in patients with cirrhosis.73 The NLRP6 inflammasome NLRP6 participates in inflammasome signalling 74 and has a critical role in infection, autoinflammation and tumorigenesis.75–78 The ligand for NLRP6 has not yet

been identified.79 Although highly expressed in neutrophils and T cells,74 the role of NLRP6 in NASH has been attributed mainly to its expression in the intestinal epithelium and role in gut physiology, which includes the modulation of gut–liver crosstalk (Figure 3).44,76–78 Data published in 2014 indicate that the NLRP6 inflammasome regulates mucus secretion from intestinal goblet cells and orchestrates the colonic host–microbial interface.80 However, the signals and mechanisms leading to NLRP6 downstream effects remain elusive. The NLRP6 inflammasome has, at present, no known direct role within the liver.

Inflammasomes in liver diseases Alcoholic liver disease Acute alcohol consumption induces hepatic steatosis and prolonged alcohol exposure leads to steatohepatitis, fibrosis and cirrhosis. The advanced stages of ALD, such as cirrhosis or severe alcoholic hepatitis, are considered irreversible, with often lethal outcomes due to liver failure.81 Activation of innate immunity and inflammation are major contributors to the progression of ALD.82 Patients with acute alcoholic hepatitis demonstrate increased serum levels of TNF, IL‑1 and IL‑8, elevated expression of CASP‑1 and NLRP3 in the liver, neutrophilia (an increase in the number of neutrophil granulocytes), and activation of monocytes and macrophages.81,83,84 Notably, patients with the most severe forms of alcoholic hepatitis have a substantial increase in serum levels of IL‑1β, compared with healthy



REVIEWS Table 1 | Experimental models of liver disease Model

Method

Study references

Alcoholic liver disease (ALD) Mouse model of chronic ALD

Lieber-DeCarli model: 2-week to 8-week feeding protocol using a liquid diet enriched with ethanol; this diet enables prolonged exposure to alcohol

17,18,95,163,164

Tsukamoto-French model: mice are implanted with a long-term gastrostomy catheter and receive ethanol infusions for 4 weeks

89,94

Mouse model of acute ALD

Administration of a single dose of ethanol via an intragastric applicator

18,160

In vitro

In vitro ethanol incubation of hepatocyte lines

26,86,87,95

Choline-deficient amino acid-defined diet

19,106

Methionine-choline-deficient diet

22,44,59,109

High-fat diet

23,25,104,152–155

High-fat diet fed to genetically obese mice

64

Hepatocytes stimulated with fatty acids

24

NAFLD/NASH Mouse models

In vitro

Paracetamol-induced liver injury Mouse model

Single intraperitoneal administration of paracetamol

45,118,121–123,126, 129,130,195–197

Ischaemia–reperfusion injury Mouse models

All structures in the portal triad (hepatic artery, portal vein and bile duct) are occluded with an atraumatic clip for 60 min and reperfusion is initiated by removal of the clamp

55,67,132,133, 137–145

Mouse models

Intraperitoneal injections of carbon tetrachloride

54,56,105,149

Administration of thioacetamide in drinking water

151

In vitro

In vitro experiments using hepatic stellate cells

146,149,150

Intravenous administration of concanavalin A Used for studies of T-cell and macrophagedependent liver injury in mice

53,57

Intraperitoneal administration of pathogenassociated molecular patterns such as bacterial lipopolysaccharide or bacterial DNA

27,158

In vitro experiments using stimulation of hepatocytes with recombinant cytokines

32

In vitro studies using human hepatoma cells

46,115,117

Liver fibrosis

Hepatitis Mouse models

In vitro Hepatitis C In vitro

Gut–microbiota interaction studies Mouse models

Dextran sulfate sodium colitis

77

Co-housing studies

77

Infection with pathogenic bacterial strains

42,80,188

individuals.84 Inflammation is, therefore, a major influence on ALD progression, and the presence of increased IL‑1β and neutrophilia, pathognomonic for sterile inflammation in absence of bacterial infection, indicates inflammasome activation.85 Likewise, the presence of Mallory-Denk bodies in liver pathology, a feature of alcoholic hepatitis, correlates with increased expression of the inflammasome components NLRP3, ASC, CASP‑1 and IL‑1β in human acute alcoholic hepatitis.83

The key role of inflammasome activation in ALD progression has been confirmed using experimental mouse models (Table 1). Chronic administration of ethanol to wild-type mice induced steatosis, liver injury and increased hepatic expression of Il‑1β, pro-Casp‑1, Asc and Nlrp3.18 Similarly, exposure of mice to ethanol increased Casp‑1 activity in the liver, indicating inflammasome activation.18 Il1r1-knockout mice and mice deficient in Casp‑1 or Asc were protected from ethanolinduced inflammasome and Il‑1β activation, and displayed attenuation of ethanol-induced liver injury and steatosis.18 The absence of inflammasome activation also prevented accumulation of inflammatory cells in the liver, resulting in the reduction of inflammatory cytokines, such as Tnf, Il‑6 and Ccl2. Daily injections of an Il-1r antagonist (Il-1ra, anakinra) ameliorated alcohol-induced liver inflammation with a dose-dependent decrease in steatosis and liver injury.18 When mice were treated with Il-1ra after 2 weeks of ethanol administration, steatosis and liver injury were also attenuated, and equally so when Il-1ra was administered during recovery from acute-on-chronic ethanol exposure (mice received the Lieber-DeCarli alcohol-containing diet for 2 weeks, followed by three daily intragastric doses of ethanol).18 Consequently, inhibition of Il‑1 signalling prevents ALD progression and expedites the recovery from alcohol withdrawal in this study. The analysis of primary mouse cells demonstrated that expression of Casp‑1, Asc and Nlrp3 is ~20-fold higher in liver immune cells than in primary hepatocytes. In further experiments, the administration of ethanol to wild-type mice induced cleavage of Casp‑1 in liver immune cells but not in hepatocytes.18 In mice with a cell-specific deletion of Casp‑1, Kupffer cells were found to be the main cell types that mediate inflammasome-dependent ALD progression.18 In another report, Nlrp3 was required for ethanol-induced activation of Casp‑1 in Kupffer cells in vitro.26 In a study published in 2014, the investigators suggested that the NLRP3 inflammasome was involved in ethanol-induced hepatocyte death.86 However, their conclusion was based on the exposure of a hepatocyte cell line to high concentrations of ethanol (1,000 mM), which were about 10 times as high as the ethanol concentration used by other investigators, thus limiting interpretation of the study.86,87 Collectively, the data available to date indicate that the pathogenic role of the inflammasome in ALD is mediated by its activation in Kupffer cells. Activators of the inflammasome in ALD have not yet been fully defined. Gut-derived lipopolysaccharide, which signals through TLR4,7,88 is likely to be the first signal that induces IL‑1β expression.18,89 Experiments using chimeric mice with cell-specific deficiency of Casp‑1 demonstrated that inflammasome activation and Il‑1β secretion in ALD is specific to Kupffer cells.18 The nature of the second activating signal that releases active IL‑1β is elusive, but alcohol-induced mitochondrial dysfunction is associated with changes in the metabolism of uric acid and ATP, two known activators of the NLRP3 inflammasome, raising the possibility that they could be the source of the activating signal.90–93 Indeed, individuals exposed to ethanol



REVIEWS have increased serum levels of uric acid,91 and treatment of alcohol-exposed rats with allopurinol, an inhibitor of uric acid synthesis, ameliorates liver inflammation, steatosis and injury.94 In our own work, we have observed a lack of alcohol-induced inflammasome activation in the livers of mice with a genetic deficiency in the ATP receptor P2X7, and in mice depleted of uric acid as a result of uricase overexpression.93 Consequently, uric acid and ATP probably represent second signals for inflammasome activation in ALD, although we cannot exclude the possibility that other host-derived molecules are also involved in this process. For example, the nonhistone chromosomal protein high mobility group protein B1 (HMGB1), an alarmin that is released predominantly from damaged hepatocytes and recognized by liver immune cells, is a strong proinflammatory signal in ALD and might activate the inflammasome.95 HMGB1 is believed to be mediated by TLR4 on the surface of liver immune cells, which leads to the induction of inflammatory cytokines.96 The investigators of a study published in 2014 indicated that HMGB1 was involved in pyroptosis of macrophages exposed to high doses of lipopolysaccharide.97 This process requires the endocytosis of HMGB1 and is dependent on CASP‑1, which indicates an interaction between HMGB1 and inflammasome signalling.97 The role of sterile signals in alcohol-induced liver inflammation is still unclear; however, current data support a role for PAMPs (derived from the gut) and DAMPs (probably derived from hepatocytes) in liver inflammation due to alcohol exposure (Figure 3). Targeting DAMPs, the inflammasome and IL‑1β might, therefore, represent promising strategies towards novel treatment models for ALD.

NAFLD The contribution of inflammasome activation in nonalcoholic liver disease conditions has received substantial interest from investigators.98,99 Much evidence has been presented that suggests inflammasome activation occurs during the development of metabolic syndrome and insulin resistance.100 Both of these conditions predispose an individual to developing NAFLD and NASH. Inflammation is thought to link obesity, metabolic syndrome, type 2 diabetes mellitus (T2DM) and NAFLD. 13,101 For example, increases in IL‑1β levels and NLPR3 inflammasome activation are associated with metabolic syndrome.25 Similarly, NLRP3 inflammasome activation was found in the blood monocytes of patients with T2DM,102 and in mouse studies islet amyloid polypeptide can induce Casp‑1 and Il‑1β production in bone‑marrow-derived dendritic cells.103 Interestingly, in animals with early NAFLD, which demonstrate liver steatosis but lack inflammation, inflammasome activation is not yet evident in the liver. As a result, tissue-specific inflammasome initiation might contribute to disease progression and an eventual expansion to other organs within the metabolic sy ndrome–NAFLD–NASH spectrum. 22,104 In early NAFLD models, mRNA upregulation of inflammasome components, such as Nlrp3, Asc and Casp1, was found in

the liver without evidence of full inflammasome activation (that is, Casp‑1 cleavage), revealing that only limited signals were available for inflammasome activation in the fatty liver.22,59,104 By contrast, investigators found evidence of increased expression of inflammasome components and activation when NAFLD had advanced to NASH.22,105 In humans with NASH, an increase in the expression of inflammasome components in the liver, such as NLRP3, ASC, CASP‑1 and pannexin, is apparent.22 Given the complex cellular environment of the liver, understanding the specificity of inflammasome activation in NASH is important. Whereas the inflammasome and its functions were initially described in immune cells and studies in alcoholic steatohepatitis suggest a specificity for Kupffer cells,18,86 the role of the NLRP3 inflammasome in NAFLD and NASH is more complex than initially predicted. In NASH, the role of the inflammasome is mediated by liver parenchymal cells in addition to liver immune cells (Figure 3).22,44,59 In the MCD-diet-induced NASH mouse model, both bone-marrow-derived cells and liver parenchymal cells contribute to inflammasome activation.59 A hepatocyte-specific role of the inflammasome in NASH is further supported by studies demonstrating that saturated fatty acids upregulate and activate the inflammasome complex in hepatocytes and induce IL‑1β production.22 To elucidate the effects of inflammasome activation on hepatocytes, some investigators have utilized mice that overexpress constitutionally active Nlrp3.105 Although the investigators were not specifically studying the pathophysiology of NASH, in this study constitutional, global activation of the NLRP3 inflammasome led to hepatocyte pyroptosis, an inflammasomedependent cell death.15 Conversely, activation of NLRP3 in myeloid cells did not result in liver pathology.105 If these findings could be replicated in a physiological model of NASH, the results would support a major pathogenic role for NLRP3 in liver parenchymal cells, including hepatocytes. In another report, the NLRP3 inflammasome seemed to be essential in NASH. Here, CDAA (choline-deficient, l‑amino acid-defined) diet induced hepatocyte death, inflammation and liver fibrosis, which was attenuated by global NLRP3 deficiency.106 The opposite effect was observed in gain-of-function tamoxifen-inducible Nlrp3 knockin mice; however, in these mice, Nlrp3 was globally expressed and not specific to any cell type.106 The inflammasome in intestinal epithelial cells can contribute to NASH by modulating the configuration of the intestinal microbiota. In a study that examined the microbiota composition and severity of fatty liver disease in mice genetically deficient in Nlrp3 or Nlrp6 inflammasomes, altering the physio logical gut microbial balance led to the appearance of members of the Prevotellaceae family and an increase in Porphyromonadaceae within the gut microflora.44,107 The size of bacterial communities from these strains was further augmented when mice were subjected to a MCD diet to induce NASH, and their abundance was associated with an increased delivery of microbial components



REVIEWS to the liver and exacerbated liver inflammation.44 A link between dysbiosis and NASH was established with the finding that antibiotic treatment ameliorated liver disease, and that the perturbed microbiota and certain aspects of the disease phenotype could be transferred to wild-type mice by cohousing them with inflammasomedeficient mice.44 Interestingly, the exacerbation of NASH seen in inflammasome-deficient mice receiving a MCD diet did not depend on inflammasomes in myeloid cells or hepatocytes. A role for inflammasomes found within alternative cell populations, probably intestinal epithelial cells, is therefore implicated in the regulation of intestinal homeostasis. Finally, according to population-based studies, NAFLD and NASH are more prevalent in men than women.108 Interestingly, inflammasomes are activated in male, but not female mice fed a high-fat diet that induces NAFLD and NASH, suggesting that sex-specific differences might regulate inflammasome activation.98,104 The ligands and triggers of inflammasome activation in NAFLD and NASH are only partially understood. Deficiencies in Myd88, Tlr2, Tlr4 and Tlr9 all attenuate NASH in mice.19,59,109 The ligands for TLR activation probably involve lipopolysaccharide from the gut microbiota and potentially other microbial danger signals, as well as TLR-activating sterile danger signals, such as HMGB1.19,59,109 The second signal in inflammasome activation is probably related to hepatocyte damage triggered by saturated fatty acids, ROS or cholesterol esters.13,23 These danger signals might accumulate during disease progression to induce, and later perpetuate, inflammation via TLRs, inflammasomes and cytokine receptors. Although the histopathological characteristics, progression to advanced liver disease and role of innate immune signalling are comparable between alcoholic steatohepatitis and NASH, the same immunopathogenic mechanisms are unlikely to be involved, which has been extensively reviewed elsewhere.110 The disease-specific differences in innate immune signalling might, at least in part, be attributable to the differential involvement of the inflammasome in alcoholic steatohepatitis compared with NASH, but additional studies are needed to provide a more comprehensive insight into this mechanism.

HCV infection In 2010, HCV infection affected >4 million people in the USA and 185 million individuals worldwide.111 HCV is sensed by multiple intracellular pattern-recognition receptors such as RIG‑I (retinoic acid-inducible gene I) or TLRs 2, 3, 4, 7/8 and 9 and typically induces production of IFN. 112,113 Among HCV-induced immune alterations, activation of monocytes and macrophages and increased proinflammatory cytokine production all contribute to the progression of HCV-induced liver disease and fibrosis.112,114 However, HCV can also activate the inflammasome complex in monocytes and macro phages and induce IL‑1β production without type I IFN induction.115 In an in vitro study in which expression of TLR3, TLR7, TLR8 or TLR9 was blocked with smallinterfering RNAs (siRNAs) in HCV, inflammasome

activation was shown to occur via TLR7.115 The involvement of NLRP3, ASC and CASP‑1 in HCV-mediated IL‑1β activation in human THP‑1 myeloid cells has also been confirmed.116 Monocyte inflammasome activation in patients infected with HCV results in IL‑18 production that in turn activates natural killer T cells.46 HCV has also been found to activate the NLRP3 inflammasome in vitro.117 Taken together, these data suggest that inflammasome activation and IL‑1β production are present in multiple cell compartments in the liver of patients infected with HCV.

Paracetamol-induced liver injury Liver injury owing to an overdose of paracetamol can be arbitrarily divided into three overlapping stages: initiation, amplification and inflammation.118 In the initi ation stage a substantial concentration of paracetamol is metabolized into the reactive metabolite N‑acetyl‑pbenzoquinone imine, which depletes glutathione, reacts with other proteins and triggers the injury process and hepatocyte necrosis. During the amplification phase mitochondrial damage is acquired and hepatocyte necrosis increases in severity.119 These two stages are crucial when predicting the survival odds of individuals with paracetamol-induced liver toxicity. In a pilot study that examined patients who took an overdose of paracetamol, markers of mitochondrial damage, which were released into the circulation during the amplification phase, helped to predict the outcome.120 During the third phase, dying hepatocytes release DAMPs that activate the inflammasome and induce inflammation in the liver.9,45 However, the effect of this final event on the extent of hepatocyte death and the outcome of paracetamol-induced liver injury remains controversial.121–123 When DAMPs are released by dying hepatocytes, DNA fragments, nuclear HMGB1, heat shock proteins and inflammasome activators, such as uric acid and ATP, are recognized by liver immune cells.9 This event is thought to be the mechanism responsible for inflammasome activation and subsequent inflammation in paracetamol-induced liver injury.124 However, whether paracetamol exposure can lead directly to the formation of the immune cell inflammasome is currently unknown. In mice, production of Il‑1β was increased in the liver in response to paracetamol,45,123 although the extent of the increase was considered disproportionally low compared with the extent of hepatocyte death.123 Specifically, serum levels of alanine aminotransferase (ALT), a marker of hepatocyte damage, showed a 500-fold increase compared with saline-treated mice, whereas expression of pro-IL‑1β in the liver increased only about fivefold.45 A lack of Il‑1β signalling in Il‑1rdeficient mice provided considerable protection from paracetamol-induced liver inflammation and hepatocyte death.45,125 However, this finding was not replicated in a subsequent study, in which a substantial dose of IL‑1β (20 μg/kg) administered after paracetamol led to the recruitment of neutrophils into the liver but did not enhance paracetamol-induced liver injury.123 Similar discrepancies were seen in studies that examined the



REVIEWS inflammasome components in paracetamol-induced liver injury. Genetic deficiencies in NLRP3, ASC, CASP1 or P2X7 provided protection from paracetamolinduced liver inflammation and injury,45,126 but these findings were not replicated in some studies.127 In fact, ATP-deficiency in P2x7-knockout mice can aggravate the extent of paracetamol-induced liver inflammation and injury.128 Conversely, depletion of uric acid in mice overexpressing uricase ameliorated paracetamol-induced inflammation but did not provide any protection from hepatocyte injury.129 These discrepancies have been attributed to multiple factors, including variability in paracetamol toxicity models in rodents, nutritional status and differences in the microbiota between mouse strains and animal facilities.9,118,121 Undoubtedly, innate immune signalling and the inflammasome are activated in paracetamol-mediated hepatotoxicity.45,130 Consequently, one can conclude that DAMPs and the inflammasome are critical determinants of paracetamol-mediated liver inflammation, but have only a limited role in the extent of hepatocyte death triggered early in injury progression by paracetamol metabolites.121–123 However, whether the inflammasome modulates liver regeneration in the recovery phase of paracetamol-mediated injury, which might be critical for the long-term outcome, is unknown. This question will probably be answered in future studies.

Ischaemia–reperfusion injury Ischaemia–reperfusion injury of the liver occurs during liver transplantation or in patients with shock due to heart failure, life-threatening bleeding or sepsis, and results from the loss of blood and reduced oxygen delivery to the liver. In the initial phase, hypoxia interferes with the function of hepatocytes and cholangio cytes, both of which are highly dependent on the oxidative metabolism. If blood and oxygen supply are restored (reperfusion), hepatocytes and cholangiocytes are exposed to oxidative stress that triggers cell death (reperfusion injury).131,132 In the clinical context, ischaemia–reperfusion injury contributes to delayed liver graft function after a liver transplantation, liver failure in patients with shock or to ischaemic cholangiopathy as a long-term consequence, resulting in secondary biliary cirrhosis.132,133 Reperfusion injury and hepatocyte death initiate the release of DAMPs, ROS production, inflammasome activation and liver infiltration with inflammatory cells.134 Notably, severe forms of ischaemia–reperfusion injury occur in livers that have been affected by a prior pathology, such as hepatocyte steatosis in liver grafts from cadaveric donors135 or in patients with pre-existing NAFLD or NASH, who have intensified ischaemic hepatitis after a septic shock.136,137 In studies using mouse models of ischaemia– reperfusion injury, Nlrp3 deficiency prevented inflammasome activation, release of Il‑1β, Il‑18 and other inflammatory cytokines, and reduced the extent of ischaemia–reperfusion injury.138,139 A similar level of protection was observed in another study using mice genetically deficient in the inflammasome components

Asc or Casp‑1,139,140 or treated with YVAD, a specific Casp‑1 inhibitor.141 These data were further supported by studies in animals in which pre-treatment with Il-1ra, delivery of Il-1ra cDNA or administration of Il‑18-neutralizing antibodies in the liver substantially reduced ischaemia–reperfusion-related liver damage, inflammation and mortality.142–144 The nature of the inflammasome-activating DAMPs in ischaemia–reperfusion injury has not been fully elucidated but some investigators suggest that histones released from dying hepatocytes activate inflammasomes in Kupffer cells, a mechanism dependent on TLR9 and ROS.139 In another study, an inhibitor of the ATP channel pannexin‑1 and an anti-cathepsin B antibody attenuated ischaemia–reperfusion-injury-induced inflammasome activation and hepatic injury,67 which suggests that ATP and crystalline material, such as uric acid or cholesterol, are involved in this context. In a study published in 2014, the investigators suggested that NLRP3 contributes to ischaemia–reperfusion injury independently of the inflammasome by regulating chemokine-mediated functions and the recruitment of neutrophils into the liver.145 To the best of our knowledge, no data have been reported regarding the direct involvement of inflammasomes in hepatocyte death due to hypoxic conditions.

Liver fibrosis Liver fibrosis can be both directly and indirectly regulated by inflammasomes. The direct pathway is mediated by inflammasome expression in HSCs, the main profibrogenic cell population in the liver.146,147 Canonical inflammasome activators, such as uric acid crystals, can activate primary mouse HSCs and human HSC lines.146 Uric acid crystals upregulate transforming growth factor (TGF)‑β1 expression, a major profibrogenic cytokine, activate HSCs, and induce production of collagen and its deposition into the extracellular matrix. These changes do not occur when HSCs lack ASC.146 The indirect pathway is thought to be mediated by HSC activation via Kupffer-cell-derived IL‑1β and IL‑18. In this scenario, gut-derived PAMPs and hepatocytederived DAMPs activate inflammasomes in Kupffer cells, and Kupffer-cell-derived IL‑1β contributes to the activation of HSCs via IL‑1β receptors.148 However, there is another inflammasome-independent mechanism that mediates cross-talk between Kupffer cells and HSCs. Specifically, liver fibrogenesis requires exposure of HSCs to gut-derived lipopolysaccharide, which downregulates BAMBI (BMP and activin membrane-bound inhibitor homolog), an inhibitory decoy receptor on the HSC surface. BAMBI-downregulation enables HSCs to respond to Kupffer cell-derived TGF‑β1 and to initiate transformation into fibroblasts.149 No experimental data supporting a role of IL‑18 in HSC activation is available. In a single study, IL‑18 did not activate HSCs in vitro.150 In experimental mouse models of liver fibrosis, Il‑1β levels are elevated and depleting Il-1r1 ameliorates this fibrotic phenotype.151 Furthermore, in rats, an absence of IL‑1 signalling attenuates dimethylnitrosamin-induced or thioacetamide-induced liver fibrogenesis.151 Similarly,



REVIEWS Condition

Dominant mechanism Metabolic

Number of known triggers

Role of the inflammasome

Immune-mediated

Autoimmune hepatitis PBC/PSC Viral hepatitis

No No Possibly

ALD NAFLD/NASH Ischaemia–reperfusion

Yes Yes Yes

Paracetamol toxicity

No Relative strength of signal

Figure 4 | Inflammasomes as systems integrators in low-signal liver diseases. Nature | Gastroenterology & Hepatology Liver diseases require both metabolic andReviews immune-mediated triggers for pathogenesis and depend on low-amplitude, repetitive signals (ALD, NAFLD or NASH) or pre-existing liver injury. In this group of diseases, inflammatory signalling is dependent on the inflammasome, which integrates multiple low-amplitude pathogenic signals and discriminates them from background noise. The inflammasome is not directly involved in diseases triggered by strong immune mechanisms (AIH, cholestatic diseases) or in disease driven by severe metabolic derangements (paracetamol-induced liver injury). Abbreviations: ALD, alcoholic liver disease; AIH, autoimmune hepatitis; PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis.

the expression of matrix metalloproteinases and tissue inhibitors of matrix metallproteinases, which are known to regulate fibrosis and tissue remodelling, is modulated by IL‑1.151 In a carbon-tetrachloride-induced or thioacetamide-induced liver fibrosis model, Tgf‑β1 and collagen‑1α1 expression was substantially reduced in mice lacking either Nlrp3 or Asc.146 The general mechanisms of fibrogenesis probably apply to the majority of chronic liver diseases; however, no direct evidence exists that supports or dismisses the involvement of the inflammasome in liver fibrosis in diseases other than ALD and NASH. For example, although ethanol is a causative agent of liver fibrosis and cirrhosis in humans, mice exposed to an ethanol-containing diet develop only a mild form of ALD. In a study using the Lieber-DeCarli ALD model, liver fibrosis was substantially reduced in Casp1-knockout mice, with a decrease in the fibrotic area in liver histology, reduced Tgf‑β1 and pro-Col1α1 expression, and reduced serum levels of procollagen III N‑terminal propeptide (PIIINP), a noninvasive fibrosis marker, tissue inhibitors of metalloproteinases‑1 and hyaluronic acid.18 In addition, administration of Il-1ra reduced serum levels of PIIINP in a dose-dependent manner. 18 Using bone marrow chimeras, the investigators found that the pathogenic effect of Il‑1 signalling relied on inflammasome activation in Kupffer cells, rather than parenchymal cells, which supports the indirect model of inflammasome-mediated liver fibrosis.18 In studies on liver fibrosis in NASH, steatosis and fibrosis were attenuated in Il-1r1-deficient mice fed with either a CDAA or high-fat diet.19,152 Similar protection against fibrosis was observed in Il‑1β-deficient mice on an atherogenic diet.153 These findings were consistent with reports in which excessive Il‑1 signalling in Il-1ra-deficient mice fed an atherogenic diet was associated with severe hepatic fat accumulation and fibrosis,

compared with wild-type controls.154 The role of inflammasome components in NASH-associated fibrosis is supported by studies that indicate a deficiency of Casp‑1 or Nlrp3 improves liver fibrosis in mice on MCD or CDAA diets, respectively.105,155 Mechanistic studies are required to elucidate whether the profibrogenic effects of inflammasome activation are the result of direct HSC activation by DAMPs or indirect DAMP-induced Kupffer cell activation with subsequent IL‑1β and IL‑18-mediated HSC activation.

Fine-tuning the inflammatory response

Liver immune cells are continually exposed to PAMPs from the external environment and DAMPs from parenchymal and nonparenchymal liver cells that undergo physiological turnover.5,9,129 Only some of these signals become potent activators of liver inflammation, which raises the question of how the hepatic immune system distinguishes between true noxious signals and benign background noise. We hypothesize that inflamma somes might mediate such discrimination, taking into account published evidence on the subject.22–26,156–159 By purposefully responding to low-threshold signals from certain PAMPs and DAMPs, inflammasomes are able to fine-tune the balance between health and liver disease (Figure 4). Pathological implications resulting from this aspect might be particularly relevant in liver diseases that are characterized by low-amplitude, repetitive signals derived from multiple sources, as in ALD, NASH, ischaemia–reperfusion injury or chronic viral infection. However, they might be less crucial in diseases in which inflammation is triggered by the primary insult itself (acute viral or autoimmune hepatitis) or by a strong metabolic impediment with a magnitude clearly exceeding background noise that is more readily detected by the immune system (such as paracetamol toxicity; Figure 4). The inflammasome might have a crucial role in mediating multiple immune response signals that can vary in their timing, cellular location and intensity, and determine the pathological outcome. For example, ethanol administration increases translocation of PAMPs from the gut to the liver and, over time, leads to hepatocyte steatosis, hepatocyte death and subsequent release of DAMPs.89–91,160–162 A single ethanol dose does not trigger any liver inflammation in mice, 160 but inflammation will develop after repeated exposure, causing repetitive increases in PAMP and DAMP levels.163,164 Consequently, the simultaneous presence of PAMPs and DAMPs might activate the inflammasome and lead to the release of IL‑1β in Kupffer cells resulting in inflammation of the liver. Indeed, in clinical observations, regular, daily alcohol intake rather than a weekly, single exposure to excessive volumes of ethanol, seems to be a strong determinant of liver inflammation in humans.165–168 The inflammasome also integrates proinflammatory stimuli in NASH, a condition characterized by the presence of repetitive but subthreshold increases in metabolic, inflammatory and endocrine signals.19,22–25,44,109,169,170 A definitive answer on whether the events described here take place in parallel or consecutively has not yet been found, and will probably be



REVIEWS elucidated in future studies. Previously published studies proposed the concept that translocation of bacterial pathogens combined with underlying steatosis are required to trigger liver inflammation in steatohepatitis (this ‘two-hit theory’ was first suggested in the context of NASH).171 However, two exposures to inflammatory signals might not be sufficient to trigger inflammation in the liver. New evidence, including our own published observations,18,22,112,158,160 favour the suggestion that signals from multiple origins, amplitude and duration are necessary for the development of liver diseases, as exemplified by the ‘multiple parallel hit hypothesis’ in the context of NASH.172

Inflammasome-targeting therapies

The central role of inflammasome activation in the pathogenesis of liver diseases makes its inhibition an attractive target for the treatment of these disorders; in particular as only a limited number of treatments is available for liver diseases. Pharmacological inhibition of the inflammasome signalling pathway can be achieved with the aid of caspase inhibitors, antibodies or endogenous IL‑1β inhibitors.

Caspase inhibitors To date, two pan-caspase inhibitors, IDN‑6556173 and PF‑03491390174, have been evaluated in human liver diseases. When administered during liver transplantation, whilst donor organs were kept in cold storage and flushed with a solution containing IDN‑6556, this drug provided 40% protection against ischaemia–reperfusion liver injury, as indicated by decreased ALT serum levels and ameliorated accumulation of neutrophils in the liver (as determined by graft biopsy) 7 days after transplantation.175 Furthermore, in patients infected with HCV, IDN‑6556 and PF‑03491390 substantially decreased serum levels of transaminases, with no reduction in HCV viral load.174,176 It is difficult to ascertain whether the beneficial effects of IDN‑6556 and PF‑03491390 in patients infected with HCV or those who had undergone liver transplantation were attributable to specific CASP‑1 inhibition or effects on proapoptotic CASP‑3 or CASP‑7, which are also targeted by these drugs. Pralnacasan, a specific CASP‑1 inhibitor, has not been tested in liver diseases.177 IL‑1 signalling inhibitors Disruption of the inflammasome activation–IL‑1β– proinflammatory cytokine cascade might also have benefits in chronic liver diseases. IL‑1 signalling can be inhibited in humans using anakinra, an endogenous inhibitor of the IL‑1 receptor, or canakinumab, a human monoclonal neutralizing antibody against IL‑1β.178 In a mouse model of ALD, anakinra attenuated liver steatosis, inflammation, hepatocellular damage and fibrosis even in the presence of continued alcohol administration.18 These findings are being evaluated for their efficacy in humans in a clinical trial assessing individuals with severe acute alcoholic hepatitis.179 IL‑1 signalling inhibition is also being tested as a treatment for patients with type 1 diabetes mellitus or T2DM.180,181 Anakinra and canakinumab were feasible and safe to use in patients

with type 1 diabetes mellitus; canakinumab also has a good safety profile in patients with T2DM.181 In addition, IL‑1 inhibition is under consideration as a treatment for NAFLD and NASH. However, whether targeting one proinflammatory signalling pathway is sufficient to attenuate tissue damage and inflammation in NASH is unclear, because multiple danger signals and receptors are involved in this process. In particular, hepatocyte damage and the resulting sterile danger signals are major contributors to inflammasome activation in chronic inflammatory liver disease.9 Consequently, interventions that prevent hepatocyte damage and death might have a benefit by eliminating sterile danger signals. In this category, caspase inhibitors are of particular interest as they can prevent cell death and/or activation of CASP‑1.182 Some drugs already in clinical use can inhibit inflammasome activation, including glyburide (used for T2DM), a specific inhibitor of the NALP3 inflammasome183 and probenecid (used for gout), a P2X7 receptor inhibitor.184 Resveratrol, a polyphenol naturally produced by grapes, can inhibit NLRP3 inflammasome activation.185 Furthermore, inhibition of heatshock protein 90, a sterile danger signal and intracellular chaperone, can attenuate lipopolysaccharideinduced liver injury in mice.186 These initial studies might yield future therapeutic interventions and strategies to limit harmful liver inflammation.

Casp‑1-deficient mice: caveats

Much of the evidence supporting the involvement of inflammasomes in liver diseases is based on studies using Casp‑1-deficient mice. However, these models were generated using embryonic stem cells that originated from a mouse strain lacking both Casp‑1 and Casp‑11.42,187 Consequently, mice described as ‘Casp‑1-deficient’ are in fact a double knockout of both Casp‑1 and Casp‑11, which might have different functionalities.42,187 For example, to dissect the roles of Casp‑1 and Casp‑11 in response to bacterial infection, Kayagaki et al.187 generated genuine Casp‑1-deficient mice, as well as a mouse knockout for Casp11 and Casp1–Casp11 double-knockout mice. In this study, Casp‑1 was required for the inflammasome response to canonical stimulators, such as ATP, monosodium urate, calcium pyrophosphate, nigericin, flagellin and poly(dA:dT), a synthetic double-stranded DNA that activates the AIM2 inflammasome.187 However, Casp‑11, but not Casp‑1, was required for inflammasomedependent cell death of bone marrow-derived macro phages that had been induced by nonc anonical inflammasome activators (such as Vibrio cholerae, Salmonella enterica subsp. enterica serovar Typhimurium [S. Typhimurium], Citrobacter rodentium, Escherichia coli, Shigella and lethal doses of lipopolysaccharides).42,187,188 Consequently, investigators who used ‘Casp1 knockout mice’ might have actually demonstrated the importance of Casp‑1 and/or Casp‑11, but not Casp‑1 alone. All the studies on liver diseases published to date did not use genuine Casp‑1 or Casp‑11-deficient mice. Should these investigations therefore be reinterpreted? Currently, evidence is lacking to support a role of Casp‑11 activators,



REVIEWS such as V. cholerae, S. Typhimurium or C. rodentium, in liver diseases. However, patients with ALD have increased serum antibody levels against E. coli,189 and it cannot be excluded that a substantial proportion of liposaccharide that was translocated from the gut to the liver 5,190 is derived from E. coli. In high concentrations, E. coli is a noncanonical inflammasome activator via Casp‑11, and Casp‑11-deficiency protects these mice against lipopolysaccharide-induced lethality.187 Kayagaki et al.187, when studying bacterial infection, used a lethal lipopolysaccharide dose that mimics a septic shock and, at such a high dose, activates the inflammasome independently of TLR4 via an unknown cytosolic mechanism.42 However, caution should be used when extrapolating these data to liver disease. In liver diseases, the increase in lipopolysaccharide levels, although important, is minor,22,88,191,192 and TLR4 signalling is required to mediate an effect.88 Although investigators who studied septic shock using ‘Casp‑1-deficient mice’ might have been misled to assume that published Casp1-knockout mice still expressed normal levels of Casp‑11,193,194 we do not see convincing evidence mandating the reinterpretation of studies in liver diseases because they are characterized by low-amplitude lipopolysaccharide release and canonical activation of the inflammasome. Studies investigating the role of Casp‑1 in Gram-negative (E. coli) sepsis-related cholestasis or liver failure, which would require the use of genuine Casp‑1‑deficient mice, have not been reported.

Conclusions

In conclusion, inflammation is a central component of most chronic liver diseases and it contributes to 1.

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