Petrasek J, Bala S, Csak T, Lippai D, Kodys K, Menashy V, et al. IL-1 receptor antagonist ameliorates inflammasome-dependent alcoholic steatohepatitis in mice. J Clin Invest 2012;122:3476-3489. (Reprinted with permission.)
Alcoholic liver disease (ALD) is characterized by steatosis and upregulation of proinflammatory cytokines, including IL-1β. IL-1β, type I IL-1 receptor (IL-1R1), and IL-1 receptor antagonist (IL-1Ra) are all important regulators of the IL-1 signaling complex, which plays a role in inflammation. Furthermore, IL-1β maturation is dependent on caspase-1 (Casp-1). Using IL-1Ra-treated mice as well as 3 mouse models deficient in regulators of IL-1β activation (Casp-1 and ASC) or signaling (IL-1R1), we found that IL-1β signaling is required for the development of alcohol-induced liver steatosis, inflammation, and injury. Increased IL-1β was due to upregulation of Casp-1 activity and inflammasome activation. The pathogenic role of IL-1 signaling in ALD was attributable to the activation of the inflammasome in BM-derived Kupffer cells. Importantly, in vivo intervention with a recombinant IL-1Ra blocked IL-1 signaling and markedly attenuated alcohol-induced liver inflammation, steatosis, and damage. Furthermore, physiological doses of IL-1β induced steatosis, increased the inflammatory and prosteatotic chemokine MCP-1 in hepatocytes, and augmented TLR4-dependent upregulation of inflammatory signaling in macrophages. In conclusion, we demonstrated that Casp-1-dependent upregulation of IL-1β and signaling mediated by IL-1R1 are crucial in ALD pathogenesis. Our findings suggest a potential role of IL-1R1 inhibition in the treatment of ALD.
Alcoholic liver disease (ALD), which affects over 140 million people worldwide, continues to be a major health concern. Acute alcohol consumption induces fatty liver and prolonged ingestion of alcohol causes progression to steatohepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma.1 The early stages of ALD are reversible with cessation from alcohol but the later stages of ALD such as cirrhosis or severe alcoholic hepatitis may not be reversible, often leading to mortality due to liver failure. There are no US Food and Drug Administration (FDA)-approved therapies for treating the later stages of ALD.2 Liver transplantation is the only option for prolonging life. Many factors have been identified that contribute to disease progression including drinking pattern, sex, nutrition, and obesity; however, despite years of extensive research, the underlying molecular mechanisms leading to disease progression are only partially understood.1 Altered immunity and inflammation are two components that have been identified as major contributors in the progression of ALD.1, 3 Patients with alcoholic hepatitis have increased serum levels of tumor necrosis factor alpha (TNF-α), interleukin 1 (IL-1), and IL-8, neutrophilia, and activation of monocytes and macrophages.1, 3 This suggests that inflammation is a major driving force in ALD progression, which has led to attempts at targeting the inflammatory pathway for potential therapeutic intervention.3 During the last two decades, the role of TNF-α in the pathogenesis of ALD received great attention because rats treated with an anti-TNF-α antibody and mice deficient in TNF-α receptor were protected from alcohol-induced liver injury.4, 5 Additionally, hepatic and serum TNF-α levels are elevated in patients with alcoholic hepatitis, and correlate with disease severity. These findings led to attempts at using TNF-α inhibitors for the treatment of ALD patients but their use was terminated due to increases in infection and mortality.6 Despite the failure of anti-TNF-α treatment in ALD patients, it is still generally believed that inflammation is a major contributor to ALD progression and targeting inflammatory pathways for potential therapeutic intervention remains an attractive strategy.3 At present, there are no FDA-approved treatments for ALD, thus discovery of novel therapies aimed at inhibiting the inflammation associated with the early stages of ALD will indeed be beneficial for slowing disease progression and improving patient outcomes.
The inflammasome is a multiprotein complex comprised of one or more NOD-like receptors (NLRs) and the pro-caspase protease capase-1 (casp-1) with or without the contribution of the adapter protein apoptosis-associated speck-like protein containing a carboxy-terminal CARD (ASC).7 To date, four types of inflammasomes have been reported: NLRP1, NLRP3, NLRC4, and AIM2. Among these inflammasomes, the NLRP3 inflammasome is the most extensively studied.7 Activation of the inflammasome leads to activation of casp-1 and subsequent maturation of IL-1 family cytokines, including IL-1β and IL-18, thereby playing important roles in host responses to microbial pathogens, cancer, metabolic, and inflammatory diseases.7 Numerous studies suggest that activation of the inflammasome contributes to the pathogenesis of various types of liver diseases.8, 9 A recent elegant study from Petrasek et al.10 revealed that activation of inflammasome-IL-1 signaling also plays a critical role in ethanol-induced liver injury in mice, suggesting the therapeutic potential of targeting the inflammasome and/or IL-1 signaling in the management of ALD. IL-1β is an inflammatory cytokine that signals through IL-1 receptor 1 (IL-1R1) leading to an inflammatory cascade that has been implicated in the progression of several types of chronic inflammatory diseases including nonalcoholic steatohepatitis.11 IL-1β, which is formed after casp-1-dependent cleavage of its precursor in the inflammasome, is significantly upregulated in ALD.10 Chronic administration of ethanol to C57BL/6 (wildtype: WT) mice induced steatosis, elevated serum alanine aminotransferase (ALT), increased hepatic expression of the messenger RNA (mRNA)s for IL-1β and the inflammasome components pro-casp-1, Asc, and Nlrp3, and increased casp-1 activity. IL-1 receptor (IL-1R) knockout (KO) mice and mice deficient in the inflammasome components casp-1 or Asc displayed attenuation of ethanol-induced liver injury, steatosis, and inflammation. Remarkably, casp-1 KO mice were also protected from ethanol-induced mild hepatic fibrosis. Together, these data suggest a critical role for IL-1 signaling in alcohol-induced liver injury, which is dependent on the formation and activation of the inflammasome. IL-1R antagonist (IL-1Ra) is a naturally occurring cytokine that binds to IL-1R1 to regulate the actions of IL-1β and control inflammation. Treatment of ethanol-fed mice with human recombinant IL-1Ra markedly reduced serum ALT and fibrosis markers, steatosis, and inflammation in the liver. Impressively, steatosis and liver injury were also attenuated in mice that received IL-1Ra after 2 weeks of ethanol feeding or when IL-1Ra was administered during cessation from alcohol following acute-on-chronic ethanol administration (4 weeks liquid diet feeding with gavage on the last 3 days), suggesting that inhibition of IL-1 signaling halts the progression of ALD and accelerates recovery following withdrawal from alcohol.
The liver is comprised of many different cell types, each containing inflammasome components8, 9 and playing their own roles in the progression of alcohol-induced liver injury; thus, it was imperative to determine the cell type(s) contributing to the pathogenesis of ALD mediated through casp-1-dependent IL-1 signaling (Fig. 1). Petrasek et al. isolated liver mononuclear cells (LMNCs) and hepatocytes from mice to determine which cell types made the most significant contribution to inflammasome-mediated liver injury. LMNCs isolated from WT mice expressed significantly higher baseline levels of casp-1 and IL-1β than isolated primary hepatocytes, and treatment with either ethanol or lipopolysaccharide (LPS) increased levels of cleaved casp-1 and IL-1β only in LMNCs. Numerous cell types constitute LMNCs, including macrophages, monocytes, T cells, and natural killer cells. Further studies from Petrasek et al.10 suggest that Kupffer cells (KCs), the resident macrophages of the liver, are the main mediators of inflammasome-dependent progression of ALD. WT and casp-1 KO mice were treated with clodronate and subjected to whole-body irradiation to remove KCs. Following KC depletion, WT mice were transplanted with bone marrow (BM) from casp-1 KO mice (WT/casp-1-KO BM) and casp-1-KO mice were transplanted with WT BM (casp-1 KO/WT BM); KC-depleted WT mice transplanted with WT BM (WT/WT BM) were used as controls. WT/casp-1-KO BM mice were protected from ethanol-induced liver injury, inflammation, and steatosis compared to WT/WT BM mice. Interestingly, casp-1 KO/WT BM mice showed slightly elevated serum ALT and steatosis compared to control mice. While these studies provide convincing evidence of the role of KCs in inflammasome-dependent progression of ALD, inflammasomes or their necessary components are found in several other cell types including biliary epithelial cells, hepatic stellate cells (HSCs), T cells, and neutrophils; each of these cells also contributes to the pathogenesis of ALD, so it is reasonable to speculate that these cell types may also play a role in, or be affected by, inflammasome-dependent IL-1 signaling. Indeed, several fibrotic markers were down-regulated in ethanol-fed casp-1 KO mice or in response to IL-1Ra treatment, but the roles of inflammasome in HSC activation were not analyzed in this study. HSCs are casp-1-expressing cells; thus, a role for casp-1 in HSC activation cannot be ruled out. Additionally, infiltration of neutrophils into hepatic tissue is a consequence of alcohol consumption and these cells play a critical role in progression of ALD; however, the role of inflammasome in these cells was also not addressed in this study.
The data presented by Petrasek et al.10 provide convincing evidence that IL-1 signaling plays an important role in ethanol-induced liver injury in mice, suggesting the therapeutic potential of IL-1 inhibitors for the treatment of ALD. At present, three IL-1 inhibitors have been approved for the treatment of several types of inflammatory diseases.12 These include the IL-1 receptor antagonist anakinra, the soluble decoy receptor rilonacept, and the neutralizing monoclonal anti-IL-1β antibody canakinumab. Additionally, a monoclonal antibody against IL-1R and a neutralizing antibody against IL-1a are in clinical trials.12 The data provided by Petrasek et al. suggest that inhibition of IL-1 signaling is beneficial for various stages of ALD, including fatty liver, steatohepatitis, and fibrosis. Because severe alcoholic hepatitis (AH) is associated with high mortality and lacks effective treatment,13 it is urgent to investigate whether inhibition of IL-1 signaling is beneficial for AH. Steroids are currently used to treat AH but their use is controversial; steroids increase short-term survival but also increase the patient's risk for infection. Compared to steroids, IL-1 inhibitors are associated with fewer adverse side effects and exhibit better safety profiles.12 Thus, it is important to determine whether IL-1 signaling is activated and contributes to the pathogenesis of AH, and whether inhibition of IL-1 signaling decreases AH-associated death and enhances patient outcomes. Further clinical studies are required to address these questions before therapeutic application of IL-1 inhibitors in patients with ALD.