Hepatitis regulation by the inflammasome signaling pathway.

Amina A. Negash Michael Gale Jr.

Hepatitis regulation by the inflammasome signaling pathway

Authors’ address Amina A. Negash1, Michael Gale Jr.1 1 Center for Innate Immunity and Immune Disease, Department of Immunology, University of Washington School of Medicine, Seattle, WA, USA.

Summary: Hepatitis is damage and inflammation of the liver. It is triggered by both environmental and endogenous insults and is a platform for developing liver cirrhosis and cancer. Both innate and adaptive immune activation contribute to hepatic inflammation and disease. Viral hepatitis is the most common form of hepatitis and is typically associated with chronic viral infection. Alcohol-induced and non-alcoholic steatohepatitis are two rising hepatic problems. The innate immune inflammasome signaling cascade mediates the production of essential proinflammatory cytokines interleukin-1b (IL-1b) and IL-18. These cytokines regulate hepatic cell interaction and crosstalk of the various inflammatory pathways and influence disease outcome.

Correspondence to: Michael Gale Jr. Department of Immunology University of Washington 750 Republican Street Seattle, WA 98109, USA Tel.: +1 206 543 8514 e-mail: [email protected] Acknowledgements A. N. and M. G. are supported in part by NIH grant AI088778. We thank Dr. Alison Kell and Lauren Aarreberg for their feedback and suggestions. The authors have no conflicts of interest to declare.

This article is part of a series of reviews covering Inflammasomes appearing in Volume 265 of Immunological Reviews.

Immunological Reviews 2015 Vol. 265: 143–155

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Immunological Reviews 0105-2896

Keywords: hepatitis, inflammasomes, proinflammatory cytokines, IL-1b and IL-18, innate immunity, inflammation

Introduction Acute and chronic hepatitis Hepatitis refers to damage and inflammation of the liver (1). The course and etiology of hepatic inflammation depends on the underlying cause, which can be triggered by microbial infection, metabolic disorders, or exposure to drugs and toxic substances. Liver inflammation can be short and self-limited (acute hepatitis), or it can be persistent and progressive (chronic hepatitis). Common clinical manifestations of acute hepatitis are jaundice, loss of appetite, dark urine, fatigue, abdominal pain, nausea, and vomiting. During this phase, the liver appears reddened and enlarged and serum levels of liver enzymes such as alanine aminotransferase (ALT) and aspartate transferase are elevated. At the microscopic level, the normal architecture of the liver is lost whereby hepatocytes become swollen, undergo necrosis, and change the rate of their proliferation (1). At this stage, hepatic inflammation is marked by massive recruitment of mononuclear cells (lymphocytes and plasma cells) and enhanced accumulation of phagocytosed debris within Kupffer cells (KCs), the resident liver macrophages. Chronic hepatitis is any form of hepatitis that shares symptoms common to acute hepatitis and shows serological

© 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

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Negash & Gale Inflammasomes in hepatic inflammation and disease

and histological (inflammation and necrosis) evidences for the presence of hepatitis for more than 6 months. At the microscopic level during chronic hepatitis, inflammation is confined to the portal tracts accompanied by fibrosis. In most cases, hepatitis is the outcome of hepatic immune cell response to environmental and/or endogenous insults, leading to tissue damage and liver pathology. Chronic hepatitis is thought to serve as a platform for onset of fibrosis and cirrhosis, underscored by progressive liver disease. Liver fibrosis is a consequence of the wound-healing response to injury (2, 3). It is characterized by massive deposition of extracellular matrix proteins, which form tough fibrous scars on regenerating hepatocytes. Hepatic stellate cells (HSCs), which are activated by infection or inflammatory cues, mediate hepatic collagen deposition. Persistent liver damage provides a suitable platform for developing nodules and promoting the onset of what is known as liver cirrhosis (1). Liver cirrhosis, which develops over the course of 10– 20 years, is the end stage of chronic hepatic inflammation, and it is thought to be a risk factor for liver failure and the onset of hepatocellular carcinoma (HCC). A cirrhotic liver displays impaired hepatic function and increased intrahepatic resistance to blood flow, which leads to portal hypertension and hepatic dysfunction, and can cause complications leading to death. The hepatic microenvironment The liver is an essential organ with a wide range of functions in the body. The liver tasks include detoxifying toxins and metabolites, metabolizing lipids, proteins and carbohydrates, and biosynthesizing essential blood proteins. Hepatocytes, which are the liver parenchymal cells, make up the majority of liver cells and carry out the bulk of hepatic metabolic functions (4, 5). Other hepatic cells include KCs, myeloid cells, HSCs, and liver sinusoidal endothelial cells (LSECs), which all reside within the sinusoidal region. As the liver macrophages, KCs play a pivotal role in sensing pathogens and triggering hepatic immune activation. These residents marophages exert their effect by producing inflammatory mediators immediately upon recognition of pathogen-associated molecular patterns (PAMPs) or dangerassociated molecular patterns (DAMPs) that present in the liver. Other hepatic cells such as dendrtic cells (DCs) and lymphocytes also play an important role in liver homeostasis and immunity. Blood from the circulation, enriched in nutrients and pathogens, enters the liver through the hepatic artery where it travels through the sinusoidal regions and

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then empties into the central vein. The tissue-wide exposure to blood renders the liver more susceptible for metabolic, microbial, and toxin insults. However, the liver has a unique architecture that allows maintenance of body metabolic homeostasis, promotion of cell-to-cell interactions, and induction of hepatic immune activation and regulation (Fig. 1). The hepatic microenvironment is tightly regulated to maintain a balance between a state of local refraction/tolerance and immunity. Any disruption in this balance due to an infection or deposition of toxic substances can lead to hepatitis, which, if left uncontrolled, could render progressive liver damage, cirrhosis, cancer, and ultimately liver failure. The interleukin-1 family cytokines: IL-1b and IL-18 Interleukin-1 (IL-1) and interleukin-18 (IL-18) are pleiotropic proinflammatory cytokines. IL-1 is an acute phase protein, which comes in two forms known as IL-1a and IL-1b (6, 7). Both IL-1a and IL-b signal through a common receptor IL-1 receptor type 1 (IL-1R1) (8, 9). IL-1 impacts a broad range of biological processes, and it is associated with inflammatory disorders (10, 11). For example, IL-1 triggers physiological changes such as fever and loss of appetite. In addition, IL-1 promotes the differentiation and maturation of monocytes and macrophages. It also modulates the activation and differentiation of T lymphocytes. IL1b has been found as a key pathologic factor in hereditary systemic autoinflammatory diseases such as familial Mediterranean fever and cryopyrin-associated periodic syndrome. Moreover, in both type-1 and type-2 diabetes, IL-1b has been implicated as toxic to b-cells (insulin-producing cells in the pancreas). Gout is an inflammatory disease causing joint pain due to accumulation of uric acid. In vitro and in vivo studies have demonstrated that uric acid activates IL-1b production (12), suggesting that IL-1b may play an important role in gout pathogenesis. Moreover, patients with osteoarthritis and rheumatoid arthritis treated with anakinra (an IL-1 receptor antagonist) show reduced inflammation, suggesting that IL-1b may be involved in the pathogenesis of these diseases. IL-18 was first known as ‘IFN-c-inducing factor’ but is now known to induce a wide range of inflammatory mediators (13, 14). As with IL-1b, macrophages and DCs are the primary producers of IL-18. IL-18 signals through IL-18Ra, which is expressed on most cells, and IL18Rb is expressed on T cells and DCs (15). IL-18 induces increased expression of adhesion molecules, nitric oxide, FasL, and chemokines. It has been implicated in the © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015


Hepatocytes

Trigger induction of proinflammatory cytokines/chemokines Or IFN induction/antagonism

DC/pDC

CD8 CD4

KC Toxic agents (Alcohol or fatty acid or drugs)

Central vein

Hepatic artery

Space of disse

IFN-γ Inflammatory mediators (IL-1β/TNF/IL-18)

Activation (Fibroblasts)

NK cell

Sinusoid region

LSECs

Fig. 1. Hepatic microenvironment. Blood is delivered to the liver through the hepatic artery then it drains into the central vein. Hepatocytes make up the majority of liver cells. Within the sinusoid region Kupffer cells (KC), hepatic stellate cells (HSCs), dendritic cells (DCs and pDCs), natural killer (NK) cells, and lymphocytes (CD4 and CD8) are found. Hepatic cells are exposed to both nutrient and potentially pathogen/toxic agent rich blood as it travels through the sinusoidal region. Infectious microbes such as hepatitis viruses can actively infect hepatocytes. KCs can respond to pathogens and/or toxic substances. They become activated to produce IL-1b and IL-18 and other inflammatory cytokines/chemokines to recruit lymphocyte and myeloid cells to the liver. DC can recognize infectious microbe and produce cytokines and chemokine in addition to priming both CD4 and CD8. HSCs become activated in response to infection or inflammatory mediators to become fibroblasts (cells involved in fibrosis). Liver sinusoidal epithelial cells (LSECs) are another cell type found within the hepatic microenvironment.

pathogenesis of mouse melanoma, as treatment of mice with anti-IL-18 showed reduction in disease, thus modeling potential of etiology, and therapy for IL-18-associated malignant disease. IL-18 synergizes with IL-12 to induce inflammatory IFN-c and other inflammatory cytokines. Further studies have demonstrated the inflammatory role of IL18 and its ability to promote pathologic IL-17 production (16) and extensive inflammatory responses (17). Inflammasome signal transduction, activation, and regulation The inflammasome is an oligomerizing, NOD-like receptor (NLR)-containing, and caspase-1-activating cytoplasmic signaling platform (18). Usually, this cytoplasmic complex is composed of an NLR, the adapter protein ASC (apoptosisassociated spec-like protein containing CARD), and the effector caspase-1 protein. Most NLRs share similar domains and have a tripartite structural arrangement (19). The C-terminal domain of NLRs is composed of a leucine-rich repeat domain, involved in sensing and autoregulation. A central domain, nucleotide-binding oligomerization domain (also known as NACHT), self-oligomerizes and binds to nucleotides or specific PAMP/DAMP signatures. The effector N-terminus region of NLRs is composed of a caspase-1 activation and recruitment domain (CARD) or PYRIN domain, which is involved in protein–protein interaction and signal © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

transduction. PYRIN-containing NLRs require ASC to associate with caspase-1 and drive active protease caspase-1 production and mature IL-1b and IL-18 secretion (20). Caspase-1 is an intracellular aspartate-specific cysteine protease, which is typically maintained within cells as an inactive zymogen (approximately 45 kd) (21–23). Once it is activated, caspase-1 undergoes proteolytic cleavage to produce the active forms (approximately 10 kd and 20 kd subunits). IL-1b and IL-18 production requires a priming step and an inflammasome complex formation step (Fig. 2). The priming step of the inflammasome, which is triggered by PAMP or DAMP recognition, drives NLR protein upregulation and inactive proIL-1b and proIL-18 production. The inflammasome assembly activating step, signal two, is triggered by products that accumulate during microbial or toxin encounter which may include ion efflux (potassium efflux), calcium signaling, danger signal (ATP), endosomal rupture (cathepsin B), protein kinase RNA-activated (PKR), cyclicdi-GMP/cyclic-di-AMP signaling, and oxidative stress (reactive oxygen species) (24–28). Several inflammasomes have been described that are induced in response to viral infections, microbial-derived components, and exposure to toxic substances. The known inflammasomes include NLRP1, NLRP3, NLRP6, AIM2, NLRC4, and RIG-I (19, 29–31). NLRP3 inflammasome is the most thoroughly studied to date. It is activated in

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Fig. 2. The inflammasome signaling pathway. Inflammasome activation requires two signals to mediate both IL-1b and IL-18 production. Signal one is initiated by PAMP or DAMP recognition to drive gene expression and inactive proIL-1b and proIL-18 expression. Inflammasome complex is composed of an NLRP, adapter protein ASC, and procaspase-1. Signal two is activated in response to diverse stimuli such as ion efflux (potassium efflux), calcium signaling, reactive oxygen species (ROS) accumulation, and lysosome rupture (cathepsin B). This activation drives inflammasome components to associate and form a macromolecular complex that mediates active caspase-1 production and subsequent maturation and secretion of IL-1b and IL-18.

between cell types such that inflammasomes can be induced and assembled in a variety of non-myeloid cells, including epithelial cells and maybe in lymphocytes (38, 39). Importantly, the liver exhibits high levels of caspase-1 relative to many tissues (40). In terms of NLR expression, early studies showed that the liver expresses NLRP1, NLRP2, NLRP3, NLRP6, NLRP 0, NLRP12, and NLRC4 at the mRNA level (41, 42). The liver parenchymal cells, hepatocytes, express caspase-1, and ASC (40, 43–46) and studies suggest that inflammasomes can be induced and activated in hepatocytes. Besides hepatocytes, inflammasomes exist in liver stellate cells (44, 47) and the sinusoidal endothelial cells (44, 48, 49). Fibroblasts, which are involved in liver fibrosis, also have been implicated to express inflammasome components (50). KCs robustly activate the NLRP3 inflammasome to produce high levels of IL-1b (46). In this review, we discuss a contemporary overview of the inflammasome in hepatic inflammation and disease. We focus on the three broad hepatitis-inducing factors: viral hepatitis, alcoholic hepatitis, and non-alcoholic fatty liver disease (NAFLD). The role of inflammasomes in liver disease

response to diverse stimuli including viral and bacterial infections, microbial-derived toxins such as pore-forming nigericin, injury-induced stress molecules such as ATP, elevated glucose levels, monosodium urate, and harmful environmental substances such as silica and asbestos (12, 32, 33). RNA viruses such as influenza virus, hepatitis C virus (HCV), and others have been shown to trigger NLRP3 inflammasome activation. The action of the NLRP3 inflammasome is suppressed by type-I IFN (IFN-I) signaling, TRIM20, miRNA-232, and nitric oxide (32, 34). The NLRP1b inflammasome is triggered in response to anthrax lethal toxin (35). The NLRC4 inflammasome is activated in response to bacterial infection such as salmonella typhimurium (36). NLRP6 is abundantly expressed in the intestine and it is important for tissue homeostasis, whereas NLRP12 inflammasome is activated in response to Yersinia pestis (37) while AIM2 inflammasome is activated in response to viral and bacterial dsDNA. Inflammasomes and their component expression in the liver It is well established that peripheral blood myeloid cells express in high abundance the inflammasome components caspase-1, NLRs, ASC, and proIL-1b/proIL-18. Moreover, specific NLR expression varies from tissue to tissue and

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Viral hepatitis Viral hepatitis is the most common form of hepatitis and describes liver inflammation caused by the major hepatitis viruses (discussed below). Although not discussed here, other viruses such as Epstein–Barr virus, lymphocytic choriomeningitis virus, dengue virus, and a range of others also cause hepatitis. The leading hepatotropic viruses that cause hepatitis are Hepatitis A virus (HAV), Hepatitis B virus (HBV), HCV, hepatitis D virus (HDV), and hepatitis E virus (HEV). Hepatitis A virus HAV typically causes a mild self-limiting acute infection, and recurrent infection is very common (51–54). Despite the presence of an effective vaccine, HAV remains a global health problem, where 1.5–3.0 million people are infected. It is easily transferred from person to person through fecaloral contamination of skin and virus exposure of mucous membranes. Once HAV reaches the gut after ingestion, it replicates in hepatocytes and is shed into bile and feces. HAV is largely endemic in developing countries where sanitation conditions are poor and clean drinking water is lacking. HAV is a small non-enveloped picornavirus that was first identified in 1973. Like other picornaviruses, HAV has © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015


a positive single-stranded RNA (ssRNA) genome that is about 7.5 kb in length. While the quality of acute hepatitis caused by each hepatitis viruses is similar, HAV-specific liver damage includes less focal necrosis, diminished KC proliferation, and reduced acidophilic bodies compared to other hepatitis viruses. Furthermore, HAV infection is not typically linked with the occurrence of steatosis which is often seen in HCV infection, and HAV infection exhibits comparably reduced necrosis and inflammation of the periportal region of the liver (52). During the symptomatic phase of HAV infection, patients experience jaundice accompanied by fever, nausea, vomiting, and fatigue. There is no specific treatment for HAV, and management of infection is usually supportive, where ample rest and intake of balanced nutrition are recommended. HAV infection can be prevented with vaccination, and effective vaccines exist both as inactivated or live-attenuated vaccine platforms. Direct evidence of inflammasome activation in HAV infection has not been reported. Nonetheless, a laboratory study of HAV-infected monkeys reported that IL-1b, IL-2, and TNF-a expression were occasionally detected in the portal inflammatory cell infiltrates during HAV infection (55). A different study reported that children with acute Hepatitis A virus infection had elevated IL-1b, TNF-a, and IL-4 at the icteric period but decreased levels during convalescence (56). A thorough investigation is needed to evaluate the mechanism of activation and action of IL-1b and IL-18 and their associated inflammasomes during HAV infection. Hepatitis B virus HBV was discovered in 1967 as a causative agent of posttransfusion hepatitis (51, 57). It is estimated that 200–300 million people are chronically infected with HBV. Among the hepatitis viruses, HBV is the only virus that harbors a circular dsDNA genome. Like HCV, HBV is transmitted through parenteral routes and acute infection often progresses to chronic infection. The two adverse outcomes of chronic HBV infection are cirrhosis and HCC. HBV infection can manifest as different states of disease ranging from high viral load with no liver disease, to inactive state (no observed hepatitis) then to active hepatitis with liver disease (57, 58). HBV infection naturally progresses through four phases where each is characterized by a distinct clinical outcome. The first, the immune tolerance phase, is considered to occur more frequently in children born to an HBV-positive mother. At this stage, patients are HBV envelope antigen (HBVeAg) positive, liver enzyme ALT level is normal, and HBV DNA is present © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

and elevated (>20 000-international unite-IU/ml), and overall the liver displays mild to no inflammation or fibrosis. The second state, the immune clearance state, features high ALT levels accompanied by inflammation without fibrosis. The third phase, chronic but immune active phase is a state where a patient could be HBeAg positive or negative. These serologic distinctions are important because HBeAg positive typically have abnormal ALT levels and HBV DNA above 2000 IU/ml, and there is active hepatic inflammation whereas antibodies to HBe protein have been linked with reduced inflammatory response as noted next. The chronic but inactive phase typically presents last, and it is a phase where a patient seroconverts from HBeAg to HBe antibody. The ALT levels are relatively normal and the liver displays improving inflammation and some resolution of fibrosis over time. This phase is typically stable in terms of disease, but the HBV inflammatory response can reactivate concomitant with virus replication and waning anti-HBV antibody response, thus leading to bouts of hepatitis that over time can compromise liver function. It is widely accepted, however, that HBV infection does not cause direct cell cytopathic effects (59). Instead the immune response to HBV infection dictates infection outcome and contributes to HBV pathogenesis. Therefore, the HBV-host interaction is a critical and complex connection that determines the extent of hepatic inflammation, liver pathology, and response to therapy (60). Myeloid cells, mainly macrophages and DCs, play an important role in HBV control. It has been shown in the HBV infection model of chimpanzees that acute HBV infection triggers transient innate immune activation that associates with clearance of the virus, with the latter dependent on T cells (60–62). Therefore, innate immune activation is critical for the initial virus control and proper priming of the adaptive immune response. Until a fully activated T-cell response commences, innate NK cells seem to play a pivotal role in controlling the virus by producing IFN-c to mediate cell killing of HBV infected cells (63). Once HBV antigen-specific cytolytic T cells (CTLs) are activated, they target infected hepatocytes that display on their surfaces HBV antigen associated with MHC-class I (64). CTLs can directly induce cell death in infected hepatocytes or by releasing an inflammatory combination of IFN-c and TNF-a to inhibit HBV replication. In this process, T-cell-mediated immune response can have three outcomes contributing to hepatitis. First, the HBV-specific T cells, after stimulation, clear the infection with no extensive liver damage (acute hepatitis). Second, T cells become activated but remain sequestered in the liver as

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tissue-resident memory T cells displaying attenuated functionality known as exhaustion (chronic infection). Third, T cells become excessively activated and induce other, nonspecific immune cell activation leading to severe hepatitis and liver inflammatory disease. Several studies have implicated the role of inflammasomes and IL-1b and IL-18 in HBV disease progression. The intrahepatic IL-1b mRNA levels were quantified in liver biopsies obtained from patients with chronic hepatitis (classified as HCV-infected only, HBV-infected only, cirrhosis, or with HCC) (65). In this report, a difference was not found in IL-1b expression among the various groups. Interestingly, they found HCV-infected livers expressed higher IL-1b compared to HBV-infected livers, suggesting that each virus employs unique mechanisms to induce/ evade innate immunity and regulate the hepatic inflammatory response. Furthermore, these observations could suggest that IL-1b may have an antiviral role during HBV infection. Indeed, treating HBV-infected hepatoma cells in culture with recombinant IL-1b exhibited anti-HBV activity (66). IL-1b treatment increased the expression of activation-induced cytidine deaminase (AID), whereas knockdown of AID abolished anti-HBV activity of IL-1b. Moreover, the HBeAg has been shown to suppress IL-18mediated NFjB signaling (67). The HBV core antigen (HBcAg), on the other hand, has been shown to induce IL-18 secretion in PBMCs (68). One study examined the role of the AIM2 inflammasome during HBV infection. In this study, PBMCs were isolated from healthy controls and from patients with either acute or chronic HBV (chronic HBV further classified as patients at immune tolerance phase, immune active phase, and inactive phase) (69). They found high levels of AIM2 is accompanied by high serum levels of IL-1b and IL-18 in acute HBV compared to chronic HBV. More specifically, during the chronic HBV infection, the immune active phase showed similar pattern of AIM2 expression and elevated serum levels of IL-1b and IL-18 as seen in acute HBV. Furthermore, they found a positive correlation between AIM2 expression and IL-1b and IL-18 levels, suggesting AIM2 and inflammasome involvement in HBV clearance. On the other hand, the levels of IL-1b and IL-18 were negatively correlated with HBV DNA and HBeAg. This study demonstrates that AIM2 is associated with an HBV-clearing immune response and that during chronic HBV this component of the immune response is likely dampened. Of interest is that type-1 interferon treatment of HBV-infected patients can lead to reduced or permanent elimination of HBV replication (70, 71). However,

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in most patients treated with IFN-a, the effect is transient, and patients show exacerbated hepatitis in the end. Williams and colleagues (71) investigated the immunomodulatory activity of interferon in HBV-infected/IFN-treated patients. In their study, they found that interferon-treated patients spontaneously had increased levels of serum TNF-a and IL1b. Yet to be delineated mechanistically, this finding raises interesting questions. Do IL-1b and TNF-a synergize with IFN to successfully eliminate HBV replication, or do they become induced by IFN treatment and remain part of the broad inflammatory response (augmenting hepatitis) in treated patients? Hepatitis C virus HCV is a global health problem. HCV is a common bloodborne pathogen that causes liver inflammation, cirrhosis, and liver cancer leading to death (72, 73). It is estimated that 2–3 million people are newly infected each year, and 170–200 million people are chronically infected. Chronic HCV infection links tightly with risk of developing liver diseases such that 5% of chronic HCV patients die of HCVrelated illnesses (74, 75). The HCV genome exhibits high sequence diversity, and there are six HCV genotypes and more than 83 subtypes (76). Genotypes 1, 2, and 3 are globally spread with genotype-1 being mostly common in the United States. Genotypes 4 and 5 are prevalent in the Middle East and Africa, whereas genotype 6 is common in Southeast Asian countries. The most common route of HCV transmission is exposure to infected blood including blood transfusion, sharing injection needles among intravenous drug users, or occupation-related exposures (77). HCV mediates an inflammatory disease where acute infection most often progresses to chronic infection characterized by persistent hepatic inflammation (78). Most importantly, persistent liver inflammation during HCV infection is thought to serve as a platform for progressive liver injury and onset of liver cirrhosis and liver cancer. HCV belongs to the hepacivirus genus and is a member of the Flaviviridea family. It is an enveloped virus carrying one copy of a positive-sense, single-stranded RNA genome. In 1989, HCV was first identified using molecular biology approaches wherein the viral genome was initially sequenced and characterized from serum. This new agent was then assigned the name ‘HCV’ as the causative agent of non-A non-B hepatitis (79). HCV RNA is 9.6 kb in length and it is flanked by highly conserved regions, the 30 and 50 non-translated regions (NTRs). HCV is a hepatotropic virus that replicates primarily in hepatocytes, although other sites © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015


of non-hepatic replication have been reported (78, 80). HCV entry requires complex interaction with several host cellular receptors including low-density lipoprotein receptor (LDLR), tetraspanin CD81, scavenger receptor class B type 1 (SR-B1), and the tight junction (TJ) proteins claudin (CLDN-1) and occludin (81–87). Initial viral attachment on hepatocytes is mediated by HCV virion interaction with low affinity LDLR, which promotes high affinity interaction of HCV virion with SR-B1 and subsequent binding to CD81. This interaction facilitates HCV virion translocation and association with TJ proteins. This sequential and complex interaction with cellular receptors and TJ proteins mediates HCV internalization through clathrin-mediated endocytosis. Once the HCV particle is released into the cytoplasm of the hepatocyte, viral RNA is exposed and HCV protein synthesis takes place from the incoming viral genome. HCV RNA is translated into a single polyprotein of approximately 3000 amino acids in length that is then cleaved by both host and viral proteases to yield three structural proteins [core, envelope-1 (E1) and envelope-2 (E-2)], six non-structural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) and an ion channel p7. Viral NS proteins are then processed and assembled into an endoplasmic reticulum-associated membrane replication complex that produces new HCV RNA. HCV replicates its RNA using the error-prone, virally encoded RNA-dependent RNA polymerase, which generates sequence-diverse subpopulations of viral genomes known as quasispecies. Besides viral factors, many host cellular factors regulate viral replication including enzymes such as phosphatidylinositol 4-kinase III a (PI4Ka), microRNAs such as micrRNA-122 (miR-122), and cellular chaperones such as cyclophilin A (cypA) (88–90). HCV is a successful virus in terms of mediating chronic infection, in part because it evades innate immune detection and its quasispecies nature allows it to evade aspects of adaptive immunity through constant antigenic drift of newly produced viruses in any given patient. Cell culture models and infection models of chimpanzees show that HCV infection can trigger type-1 interferon production minutes to hours after recognition of the viral infection by host cells. However, through the action of its protease, NS3/4A, HCV can target and cleave mitochondrial antiviral-signaling (MAVS) protein, the essential adapter molecule for RIG-Ilike receptor signaling, to block interferon induction and thus support progression from acute to chronic infection (91–93). Other innate immune evasion mechanisms further support viral persistence, including NS3/4A blocking of © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

TLR3 signaling, viral interference with interferon signaling, and other features of innate immune control (93). Acute hepatitis during HCV infection is characterized by robust innate immune activation and potent CTL activity leading to virus control and clearance in the best cases. On the other hand, dysregulated inflammatory cytokine production and attenuated T-cell responses are the key features of chronic hepatitis from HCV infection. Importantly, inflammasome-derived IL-1b and IL-18 are central regulators of hepatic inflammation. Patients with chronic hepatitis C have elevated levels of IL-1b compared to healthy controls, and intrahepatic mRNA of IL-1b expression is found to associate with severe liver disease (cirrhosis) (46, 65, 94). While HCV attenuates innate antiviral immune defenses of the infected hepatocyte, it targets a different cell to trigger inflammatory IL-1b. Of note is that monocytes and macrophages do not support HCV replication. Instead, through passive uptake or via receptor-mediated endocytosis, HCV stimulates robust IL-1b production both in macrophages and monocytes derived from healthy (treated with HCV ex vivo) or HCV-infected PBMCs (46, 65, 95). In particular, THP-1 cells were established as an in vitro model of macrophages or Kupffer cells exposed to HCV. Examination of this model revealed the mechanism of HCV-induced IL-1b production in which HCV drives rapid caspase-1 activation and IL-1b production in a manner dependent on NLRP3 and TLR7/MyD88 signaling. Hepatocytes express inflammasome components, but their ability to produce secreted IL-1b when infected appears to be dampened through virus-directed mechanisms or through host-mediated processes that control hepatocyte responses to inflammatory stimuli (46). Thus, at least in culture, HCV-infected hepatocytes do not produce IL-1b to the same quality and quantity observed in HCV-exposed macrophages. One study reported a very low level of IL-1b produced from hepatoma cells after 4 days of infection. While it is agreed upon that macrophages are the major IL-1b-producing cells in response to HCV, one cannot discount the role of hepatocytes or other hepatic cell-derived IL-1b in vivo. How IL-1b produced by each hepatic cell contributes to the overall hepatic inflammation and HCV pathogenesis requires further investigation. IL-1b production has been shown to associate with HCV related disease and HCC (65, 96–98). In a similar fashion, IL-18 has been implicated in HCV pathogenesis. Patients with acute HCV infection express high serum levels of IL-18 (99). Furthermore, monocytes exposed to HCV produce IL-18, which activates and triggers IFN-c production by natural killer cells (100).

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In many instances IL-18 has been correlated with liver injury during HCV infection (101–104). Hepatitis D virus HDV, also known as the delta agent or delta hepatitis, is linked with the most severe form of all viral hepatitis (105, 106). HDV is a unique negative sense ssRNA virus or viroid, so called because it is a non-functional virus unless paired with HBV. In this sense, HDV is dependent on HBV surface antigen to assemble its envelope and thus form new virions that can be transmitted and continue infection. HDV infectivity is thus dependent on the HDV particle being coated with hepatitis B surface antigen (HBsAg). HDV is a worldwide problem that causes infection in an existing HBV infection. It is transmitted along with HBV through infected blood, percutaneous, and permucosal (sexual) routes. HDV, depending on the infection type with HBV, can cause acute or chronic infection. Acute infection with HDV occurs due to exposure to serum infected with both HBV and HDV. This causes simultaneous acute HDV and HBV infections, and most patients clear the viruses. Chronic HDV infection, in contrast, occurs during superinfection whereby an already chronic HBV infected individual is subsequently exposed to HDV. This form of HDV infection is more severe, leading to accelerated hepatitis, chronic active hepatitis, and cirrhosis. Most of the drugs administered to treat other hepatic viruses or drugs that work against HBV do not work for suppression of HDV. The only therapy for HDV is pegylated interferon-a-based therapy, but most patients relapse after therapy cessation. HDV, like other hepatic viruses, is not cytopathic, but the liver damage and pathology associated with it is thought to be immune-mediated. Large delta hepatitis antigen (LDHAg) is encoded by HDV and has been shown to modulate NFjB signaling in infected hepatocytes and is thus implicated in HDV pathogenesis (107, 108). Inflammatory combinations of IFN-c and IL-12 are produced to high level in HDV-infected patients and are modulated with interferon treatment (109). Although the magnitude of inflammatory cytokines such as IL-1b and IL-18 and the exact molecular mechanism by which they influence hepatic damage during HDV infection remains to be delineated, one can speculate that the accelerated onset of severe hepatitis during HDV infection could be due to rapid innate immune cell activation and rampant production of key inflammatory mediators that lead to increased cell

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recruitment and massive induction of CTL activity leading to liver inflammation and disease. Hepatitis E virus HEV and HAV share many similar features. Like HAV, HEV causes a self-limited acute infection (110, 111). However, pregnant women are at high risk of liver disease and death due to HEV infection. HEV is transmitted through fecal-oral spread by exposure to contaminated water and is prevalent in developing regions of the world. Hepatitis associated with HEV is a continuing problem where close to 14 million people are impacted. HEV is a non-enveloped virus that belongs to the Hepeviridae family and harbors a ssRNA genome. HEV-induced pathology is similar to other forms of viral hepatitis, but what role inflammasomes and inflammatory cytokine signaling play in HEV-induced hepatitis is unknown. Further research is needed to uncover the role of inflammasomes, IL-1b, and IL-18 during HEV infection. Non-alcoholic fatty liver disease Non-alcoholic fatty liver disease is a metabolic syndrome where hepatitis associated with it is a growing epidemic. Non-alcoholic steatohepatitis (NASH) is the most common liver disease and it is highly prevalent in the western world (112). In the United States, NAFLD accounts for 75% of chronic liver disease (112). NAFLD is disease of all ages and fatty liver disease progresses with age. NASH was first described in 1980 (113) as a disease where lipid deposits appear within the liver in the absence of alcohol consumption. The spectrum of NAFLD disease progression begins with a very benign steatosis, which is defined by the presence of lipid within hepatocytes, then progresses to NASH and cirrhosis. Steatosis is characterized by hepatocyte damage along with inflammation. Progression from steatosis to NASH is associated with worse clinical outcomes. Approximately, 10–30% of patients with NASH will develop cirrhosis over the course of a decade. The exact disease etiology of NASH is not fully understood, but several factors have been attributed to contribute to NASH pathogenesis such as host genetics, inflammation, metabolic pathways, and microbiota. Key cells that mediate NASH development are the KCs and HSCs (114). Many inflammatory mediators produced by KCs have been implicated in the pathogenesis of NASH. TNF-a has been associated with the progression from steatosis to NASH (115). Plasma and intrahepatic TNF-a correlate with increased inflammation, steatosis and histological evidence of liver damage in patients with NASH (116). © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015


According to a study of resveratrol (117), administration of this drug to mice led to the modulation of the NLRP3 inflammasome and consequent protection of mice from hepatic inflammation, insulin-resistance and type-2 diabetes, thus implicating the NLRP3 inflammasome in NASH pathogenesis. The association of inflammasomes with NASH has been reported to be profibrotic. Hence, inflammasomes are thought to be part of the pathway linking NASH pathogenesis to liver fibrosis and progressive liver disease. Many studies have demonstrated that the inflammasome and expression of its components correlate with NASH. Saturated and mono-saturated free fatty acids are abundantly found in NAFLD patients (118). Mice fed high-fat diet (methionine-choline deficient diet) showed IL-1b production both in the serum and liver (43). NLRP3 and NLRX1 activation were shown to promote NASH pathogenesis (119, 120). Loss-of-function studies using NLRP3 knockout mice revealed that mice had reduced inflammation and were protected from severe fibrosis. In contrast, in gain of function studies using the knock-in approach, persistent NLRP3 expression was associated with proinflammatory cytokine and chemokine production, a hyper-inflammatory hepatic state and severe fibrosis. Comparing NAFLD patients with and without NASH revealed that NASH patients display elevated expression of NLRP3, IL-1b, and IL18 in the liver and each correlated with fibrosis. On the other hand, genetic deficiency of caspase-1 has been shown to ameliorate high-fat diet-induced hepatitis and fibrosis in mice (121, 122). Importantly, lysosomes are known to mediate palmitic acid-induced NLRP3 inflammasome activation in macrophages (123). A study in mice showed that Kupffer cells and HSCs are stimulated in response to either toll-like receptor-2 (TLR2) or palmitic acid to drive the initial priming signal of inflammasome activation (124, 125). Moreover, the study found that wildtype mice exhibit profound and severe inflammation and NASH when fed highfat diet compared to TLR2-deficient mice. The process of NASH development coincided with high NLRP3 expression and increase in serum IL-1b and IL-18 levels. Another TLR that is implicated in NASH is TLR9 (126). TLR9-deficient mice when fed high-fat diet showed reduced steatohepatitis and fibrosis and had suppressed expression of IL-1b. In addition, a different study showed that IL-1b treatment of hepatocytes and HSC stimulated lipid deposition and fibrogenic activity in these cells, respectively. Also absence of both IL-1b and IL-1a prevented progression from steatosis to steatohepatitis (127). Further demonstrating the © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Immunological Reviews 265/2015

pathologic role of inflammasomes in NASH, mice with NLRP6 and NLRP3 inflammasomes promoted NASH progression, demonstrating a deleterious relationship between NLRP3/NLRP6 inflammasomes and NASH that is mediated through modulating the gut microbiota (128). Alcohol liver disease Alcohol is the most widely used substance that causes toxic liver injury. Excessive alcohol consumption leads to liver damage and enhances other liver-associated disease including viral hepatitis and NAFLD. Alcohol liver disease (ALD) is a global burden (129, 130). According to the World Health Organization, two billion people consume alcohol and of those, 76.3 million people have alcohol-related disorders (131). ALD is defined as a range of disorders including steatosis, inflammation, and steatohepatitis. Prolonged alcohol intake leads to hepatic inflammation and injury, hence the name alcoholic hepatitis. Individuals with alcoholic hepatitis are at risk of developing fibrosis and cirrhosis. More than 90% of heavy alcohol drinkers have steatosis where 20–40% with steatosis develop fibrosis, of which 10–20% of them progress to cirrhosis. Alcohol consumption has been shown to drive expression of inflammatory mediators such as IL-1, TNF-a, and TFG-b. Moreover, alcohol has been reported to increase gut permeability (132), leading to LPS relocation into the portal circulation, which upon recognition by KCs triggers inflammatory cytokine production. Alcohol promotes collagen accumulation and it has been correlated with inhibition of anti-fibrotic action of NK cells (133, 134). NK-derived IFN-c induces HSC cell cycle arrest and apoptosis. Although it is well established that inflammatory mediators contribute to ALD pathogenesis, the exact role of inflammasomes in ALD is still unclear. When mice deficient in NLRP3 protein are exposed to continuous alcohol diet, they developed severe liver injury, high plasma ALT and IL18 levels, and reduced IL-1b, suggesting a protective role of NLRP3 in alcohol-induced liver injury (135). In a similar way, NLRC4-deficient mice had reduced IL-1b production when subjected to chronic alcohol consumption. However, a separate study showed in the absence of ASC or caspase-1, alcohol-fed mice exhibited reduced liver damage and attenuated upregulation of IL-1b, TNF-a, and IL-16, suggesting a pathologic role of ASC and caspase-1 (136). Furthermore, blocking IL-1b activity through pharmacological intervention with recombinant IL-1RA (anakinra) ameliorated alcohol-induced liver inflammation and damage. Polymorphisms in the IL-1b gene have been correlated with alcoholic hepatitis (137). Furthermore, elevated IL-18 expression has been

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associated with advanced alcoholic liver disease (alcoholic cirrhosis) (138). Systematic or systems biology-focused investigations are needed to delineate the exact alcoholresponsive cells in vivo and reveal the role of inflammasome components associated with ALD. Conclusion and perspectives Hepatitis is the outcome of a complex interaction of hepatic cells where inflammatory mediators orchestrate interactions and regulate intrahepatic inflammatory pathways. Great progress has been made in understanding the molecular basis of inflammation. Notably, the characterization of the inflammasome pathway and its signal transduction processes has provided new insights to define the mechanisms of inflammatory IL-1b and IL-18 production and their link to hepatitis (Fig. 3). As discussed in this review, IL-1b and IL-18 production could positively or negatively impact hepatic inflammation and disease outcome as revealed in several studies. Many questions therefore remain unanswered: what is a stimulus-specific threshold of IL-1b and IL-18 that is required to promote a protective versus a pathologic hepatic inflammatory outcome? Importantly, what is the in vivo responsive cell that produces IL-1b and/or IL-18 when the

liver is exposed to microbial infection and/or toxic agents? Similarly, what is the nature of signaling crosstalk between IL-1b and IL-18 signaling and other intrahepatic inflammatory cytokine pathways such as type-1 interferon, IFN-c, TNF-a, and the various chemokines associated with hepatic inflammation that control the outcome of inflammatory disease? While the role of inflammasomes is now appreciated in both chronic HBV and HCV infection, further research is needed to investigate inflammasomes in both HDV and HEV-associated hepatitis. Moreover, additional research is needed to unravel the role of inflammasomes and their components in ALD. NASH is a rising problem where IL-1b and IL-18 mediate liver inflammation and underlie liver pathogenesis. One exciting consideration here is whether or not anti-IL-1b/anti-IL-18 can be used as therapies to alleviate hepatic inflammation and disease. In the case of HBV, IL-1b appears to exhibit anti-HBV activity, although further investigation is needed to validate this point. Could these observations suggest that HBV is a weak inducer of IL-1b, or does HBV target the inflammasome pathway to shut down IL-1b production in order to cause persistent infection? Type-1 interferon has been shown to negatively

HCV HCV IL-1β ?

Hepatocytes

?

IL-1β/IL-18

NK cell

Alcohol Or fatty acid

Hepatitis viruses

IFN-α/β

Unkno wn tar ge

t cell

HCV V

IL-1β/IL-18

? Hepatic macrophages

?

DC/pDC

IL-18 CD4 CD8

IFN-γ LSECss

Fig. 3. Proinflammatory IL-1b and IL-18 are regulators of hepatitis. Both IL-1b and IL-18 are produced in response to infection by hepatitis viruses, alcohol intake, and fatty acid accumulation. Hepatitis viruses such as HCV and HBV induce IL-1b production, while further research is required to delineate the role of IL-1b and/or IL-18 in HAV, HDV, and HEV infection. HCV infects hepatocytes and attenuates IFN signaling within them to support viral persistence. Although hepatocytes can express intracellular IL-1b during HCV infection it is unknown whether or not HCV-infected hepatocytes in vivo secrete IL-1b. Hepatic macrophages produce IL-1b during HCV infection. Alcohol induces IL-1b and/or IL-18 production. However, the alcohol-responsive cells and the mechanism of inflammasome signal one and two induction requires further research to fully define actions on liver inflammation outcome. IL-1b plays important role in fatty acid-induced liver pathology. The mechanism of how fatty acid induces IL-1b and IL-18 remains to be delineated. It is also unclear if hepatic marophages are directly modulated by alcohol or fatty acid in vivo to produce both inflammatory cytokines and chemokines. IL-1b and IL-18 presence within the liver and their signaling cross-talk with IFN and other mediators could modulate hepatic cell activity such as hepatocytes, HSC, and LSEC.

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regulate IL-1b/NLRP3 inflammasome. Therefore, it is important to decipher the regulation of interferon-inflammasome cross-talk. This is especially critical for patients who fail to respond to interferon therapy in viral hepatitis infections and treatment. The inflammasome pathway plays

a central role in hepatitis, and understanding how hepatic inflammasomes are activated and/or regulated could offer new approaches for the development of effective antiviral therapy, immune-modifying treatments, and preventative or therapeutic hepatitis vaccines.

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Non-transcriptional regulation of NLRP3 inflammasome signaling by IL-4.

Regulation of PCP by the Fat signaling pathway.

Modulation of the Inflammasome Signaling Pathway by Enteropathogenic and Enterohemorrhagic Escherichia coli.

Regulation of retinal angiogenesis by phospholipase C-β3 signaling pathway.

Regulation of Nlrp3 inflammasome by dietary metabolites.

Regulation of inflammasome activation.

RNA viruses promote activation of the NLRP3 inflammasome through a RIP1-RIP3-DRP1 signaling pathway.

T-lymphocyte Kv1.3 channel activation triggers the NLRP3 inflammasome signaling pathway in hypertensive patients.

Molecular and chemical regulation of the Keap1-Nrf2 signaling pathway.

Regulation of insect behavior via the insulin-signaling pathway.

BMAL1-dependent regulation of the mTOR signaling pathway delays aging.

The PPARα-FGF21 hormone axis contributes to metabolic regulation by the hepatic JNK signaling pathway.

Curcumin Ameliorates Diabetic Nephropathy by Suppressing NLRP3 Inflammasome Signaling.

Regulation of global and specific mRNA translation by the mTOR signaling pathway.

Regulation of Schistosoma mansoni development and reproduction by the mitogen-activated protein kinase signaling pathway.

β-Catenin Signaling Pathway by Human Papillomavirus E6 and E7 Oncoproteins.

Lactobacillus helveticus SBT2171 inhibits lymphocyte proliferation by regulation of the JNK signaling pathway.

Regulation of stem-like cancer cells by glutamine through β-catenin pathway mediated by redox signaling.

The Regulation of Lipid Deposition by Insulin in Goose Liver Cells Is Mediated by the PI3K-AKT-mTOR Signaling Pathway.

Role of calcium in regulation of phosphoinositide signaling pathway.

Inhibition Regulation Signaling Pathway.

Autophagy regulation by nutrient signaling.

Slug contributes to cancer progression by direct regulation of ERα signaling pathway.

Regulation of PI3K by PKC and MARCKS: Single-Molecule Analysis of a Reconstituted Signaling Pathway.