The Intestinal Microbiota in Chronic Liver Disease

CHAPTER THREE

The Intestinal Microbiota in Chronic Liver Disease Jorge Henao-Mejia*,1, Eran Elinav†,1, Christoph A. Thaiss†,1, Richard A. Flavell*,‡,2

*Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA † Immunology Department, Weizmann Institute of Science, Rehovot, Israel ‡ Howard Hughes Medical Institute, Chevy Chase, Maryland, USA 1 Equal contributors 2 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Role of the Intestinal Microbiota on Chronic Liver Diseases 2.1 Nonalcoholic fatty liver disease 2.2 Cirrhosis and associated comorbidities 2.3 Hepatocellular carcinoma 2.4 Autoimmune liver disease 3. Role of the Interactions Between the Innate Immune System and the Intestinal Microbiota on Chronic Liver Diseases 3.1 Toll-like receptors 3.2 Inflammasomes 3.3 C-type lectins 3.4 Dysbiosis associated with innate immune deficiency and its implications for liver disease 4. Probiotics and their Potential Role in Liver Disease Therapy 5. Conclusions References

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Abstract Recent evidence indicates that the intestinal microflora plays a critical role in physiological and pathological processes; in particular, it is now considered a key determinant of immune pathologies and metabolic syndrome. Receiving the majority of its blood supply from the portal vein, the liver represents the first line of defense against food antigens, toxins, microbial-derived products, and microorganisms. Moreover, the liver is critically positioned to integrate metabolic outcomes with nutrient intake. To accomplish this function, the liver is equipped with a broad array of immune networks. It is now evident that, during pathological processes associated with obesity, alcohol-intake, or autoimmunity, the interaction between these immune cell populations and the

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intestinal microbiota promotes chronic liver disease progression and therefore they represent a novel therapeutic target. Herein, we highlight recent studies that have shed new light on the relationship between the microbiome, the innate immune system, and chronic liver disease progression.

1. INTRODUCTION The human gastrointestinal tract contains 10–100 trillion bacteria and approximately 500–1500 different bacterial species (Lozupone, Stombaugh, Gordon, Jansson, & Knight, 2012). These microorganisms have critical functions in multiple aspects of human physiology such as regulation of metabolic processes, education of the immune system, and promotion of epithelial cell responses that are essential to maintain mutualism (Maynard, Elson, Hatton, & Weaver, 2012; Tremaroli & Backhed, 2012). The intestinal microflora differs quantitatively and qualitatively among species and individuals. Life style, age, dietary habits, exposure to antibiotics, and host genotype play essential roles in the composition of the intestinal microflora (Claesson et al., 2012; Turnbaugh et al., 2009); moreover, disruption of the delicate balance that represents the ecosystem of bacterial communities of the gastrointestinal tract can lead to severe metabolic and inflammatory pathologies. The close functional relationship between the liver and the gastrointestinal tract (gut–liver axis) is highlighted by multiple important physiological processes that intimately interconnect these organs. The liver, the largest organ in the body, has a dual blood supply. The hepatic artery, which arises from the celiac artery, supplies oxygenated blood to the liver, and the portal vein conducts venous blood from the intestines and the spleen. Approximately 75% of hepatic blood flow is derived from the hepatic portal vein (1000–1200 mL/min), and therefore, the liver is constantly exposed to nutrients, toxins, food-derived antigens, microbial products, and microorganisms derived from the intestinal tract (Miyake & Yamamoto, 2013). This strategic location confers critical metabolic, immunologic, and detoxifying roles to the liver and stresses the crucial role of the intestinal microbiota on hepatic pathophysiology. In this review, we examine the impact of gut microbiota on hepatic diseases, focusing on how dysbiosis and immune responses triggered by microbiotaderived products shape the progression of chronic liver pathologies.

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2. ROLE OF THE INTESTINAL MICROBIOTA ON CHRONIC LIVER DISEASES 2.1. Nonalcoholic fatty liver disease Nonalcoholic fatty liver disease (NAFLD) is the leading cause of chronic liver disease in Western societies, with a prevalence ranging from 20% to 40% in the general population and up to 75–100% in obese individuals (Ludwig, Viggiano, McGill, & Oh, 1980; Sheth, Gordon, & Chopra, 1997). NAFLD is considered the hepatic manifestation of metabolic syndrome (Marchesini et al., 2003), with many patients developing other comorbidities including insulin resistance, hyperlipidemia, cardiovascular disease, polycystic ovary syndrome, and obstructive sleep apnea (Cerda et al., 2007; Tolman, Fonseca, Dalpiaz, & Tan, 2007). While most patients with NAFLD remain asymptomatic, 20% progress to develop chronic hepatic inflammation (nonalcoholic steatohepatitis, NASH), which in turn can lead to cirrhosis, portal hypertension, hepatocellular carcinoma (HCC), and increased mortality (Caldwell et al., 1999; Propst, Propst, Judmaier, & Vogel, 1995; Shimada et al., 2002). NASH can be classified as primary NASH (associated with obesity, type 2 diabetes (T2DM), and hyperlipemia) and secondary NASH (occurring after pharmacological interventions, parenteral nutrition, jejunoileal bypass surgery, or Wilson’s disease). Despite its high prevalence, factors leading to progression from NAFLD to NASH remain poorly understood and no treatment has proved effective (Charlton, 2008; Hjelkrem, Torres, & Harrison, 2008). A “two-hit” mechanism is proposed to drive NAFLD/NASH pathogenesis (Day & James, 1998). The first hit, hepatic steatosis, is closely associated with lipotoxicity-induced mitochondrial abnormalities that predispose the liver to additional proinflammatory insults (second hits) that promote disease progression. Second hits include increased generation of reactive oxygen species, increased lipid peroxidation, and gut-derived factors. Most likely, the parallel action of these hepatic tissue insults is required for the development of steatohepatitis (Sanyal et al., 2001). In the past decade, a growing body of research functionally links the intestinal microbiota with the development of steatosis (first hit) and with the progression to NASH (second hit). Obesity is considered the most common risk factor for NAFLD in humans (Younossi et al., 2011). Several lines of evidence unequivocally link the intestinal microflora with body weight and body fat composition

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Type 2 diabetes • Endotoxemia • Insulitis • Insulin resistance Intestinal microbiota

Steatosis Obesity • Increased calorie extraction • Cleavage of dietary polysaccharides • Dyslipidemia

Decreased choline metabolism

Figure 3.1 Effects of the intestinal microbiota on the risk factors that promote NAFLD development. The microbiota can regulate the progression of multiple associated comorbidities that are associated with NAFLD pathogenesis such as choline metabolism, obesity, and diabetes mellitus.

(Fig. 3.1). In animal studies, germ-free mice have a lower body fat content than conventionally raised mice; moreover, the inoculation of germ-free mice with microbiota from wild-type mice results in a significant increase in body fat accumulation (Turnbaugh et al., 2006). The phyla Bacteroidetes and Firmicutes represent a large proportion of the intestinal microbiota composition in mice and humans; however, their relative abundance profoundly affects the body composition of individuals (Ley et al., 2005; Ley, Turnbaugh, Klein, & Gordon, 2006). Genetically obese mice (ob/ob) have a significant increase in the Bacteroidetes to Firmicutes ratio when compared with lean littermate controls, but perhaps more importantly, germ-free mice colonized with microbiota from genetically obese mice gained weight faster and harvest calories more efficiently than mice colonized with intestinal microflora from lean mice (Turnbaugh et al., 2006). These findings indicate that the composition of the microbiota directly influences calorie extraction, body fat composition, and body weight. In humans, several lines of evidence now correlate the composition of the intestinal microbiota with multiple metabolic and inflammatory parameters as well as dietary habits (Claesson et al., 2012; Ley et al., 2006; Muegge et al., 2011). Similar to mice, obese individuals have increased levels of Bacteroidetes and the reduction of this phylum in the intestinal microflora is significantly associated with weight loss either by

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fat- or carbohydrate-restricted diets, suggesting that Bacteroidetes may be responsive to calorie intake (Ley et al., 2006). Metagenome-wide association studies have recently demonstrated that T2DM patients are characterized by gut microbial dysbiosis, a decrease in the abundance of butyrate-producing bacteria and an increase in various opportunistic bacterial pathogens. Moreover, these gut microbial markers can be useful for classifying T2DM, indicating that specific conformations of the intestinal microbiota play critical roles in the pathogenesis of T2DM and associated disorders (Qin et al., 2012). Calorie intake of Western society diets is a key determinant of metabolic syndrome. Long-term dietary habits have a profound effect on the human gut microbiota and therefore on potential deleterious metabolic outcomes. It has been proposed that the human gut microbiota should be divided into three compositions (enterotypes), yet this notion is still debated and merits further validation. Each suggested enterotype is dominated by a different genus—Bacteroides, Prevotella, or Ruminococcus—(Arumugam et al., 2011). Interestingly, enterotypes dominated by Bacteroides are associated with diet rich in protein and animal fat (Western diet), while Prevotelladominated enterotypes are associated with the consumption of a diet rich in carbohydrates/fiber (De Filippo et al., 2010; Wu et al., 2011), suggesting that the gut microbiota is shaped by the different diets to maximize energy extraction. Taken together, these studies show that the composition of the microbiota is a critical player in the metabolic status of the host and its disturbance is associated with metabolic abnormalities that are associated with the “first hit” (steatosis) during NAFLD pathogenesis. Although it is now clear that the intestinal microflora plays critical roles in body fat accumulation and weight gain, the role of gut-derived factors on NAFLD progression has just begun to be elucidated. Progression from steatosis to steatohepatitis is mainly an inflammatory process that likely reflects the concerted deleterious effects of multiple noxious stimuli. Several lines of evidence now suggest that intestinal bacterial communities might play an important part in this process. Jejunoileal bypass, small intestinal diverticulosis, total parenteral nutrition, and intestinal failure are associated with NASH progression (Carter & Karpen, 2007; Corrodi, 1984; Nazim, Stamp, & Hodgson, 1989; Quigley, Marsh, Shaffer, & Markin, 1993; Vanderhoof, Tuma, Antonson, & Sorrell, 1982); interestingly, small intestinal bacterial outgrowth (SIBO) as a consequence of low intestinal motility has been proposed as a key determinant factor for NAFLD progression in these conditions in humans (Carter & Karpen, 2007; Pappo et al., 1992; Quigley et al., 1993). In concordance with this, antibiotic treatment or

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surgical removal of the bypassed section of the intestine reverses SIBO and steatohepatitis. Similarly, rats fed under total parenteral nutrition are characterized by severe liver injury secondary to bowel hypomotility, which leads to the expansion of Gram-negative bacterial populations and increased hepatotoxic mediators such as endotoxin or tumor necrosis factor (Pappo et al., 1992). The role of the intestinal microbiota in the more highly prevalent primary NASH is less clear. The prevalence of SIBO is significantly increased in obese individuals as compared with healthy lean subjects (Sabate et al., 2008), but its role in NAFLD progression has largely been overlooked. Nevertheless, a recent study conducted by Miele et al. (2009) evaluated intestinal permeability, SIBO, and NAFLD disease stage. Interestingly, patients with NAFLD were reported to have significantly increased gut permeability and SIBO when compared with healthy individuals, suggesting that overgrowth of the intestinal bacterial flora gut could lead to bacterial translocation, portal endotoxemia, and ultimately hepatic injury (Miele et al., 2009). In concordance with this possibility, multiple studies have found high levels of SIBO prevalence in different cohorts of NASH patients (Sajjad et al., 2005; Wigg et al., 2001); moreover, we recently demonstrated that inflammasomemediated dysbiosis characterized by an expansion of the Prevotellaceae and Porphyromonadaceae families as well as the TM7 taxa promotes NAFLD progression in different mouse models (Henao-Mejia, Elinav, Jin, et al., 2012). Collectively, these studies indicate that different compositions of the bacterial communities of the intestines might regulate NAFLD progression in humans and therefore represent a novel therapeutic target. Characterization of the bacterial communities at different stages of NAFLD and the exact role of metabolites derived from the bacterial microflora in disease progression should shed some light on the precise role of the microbiome in liver disease in the context of metabolic syndrome.

2.2. Cirrhosis and associated comorbidities Cirrhosis is the final clinical–histopathological stage of a wide array of liver diseases. The intestinal microbiota is a common denominator of the major complications of liver cirrhosis, including spontaneous bacterial peritonitis, hepatic encephalopathy (HE), and esophageal variceal bleeding (Basile & Jones, 1997; Campillo et al., 1999; Guarner & Soriano, 1997; Husova et al., 2005; Thalheimer, Triantos, Samonakis, Patch, & Burroughs, 2005). The process of liver fibrogenesis promotes dysbiosis and intestinal

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barrier dysfunction through multiple pathological processes. Cirrhotic patients have decreased blood flow through the portal vein and intestinal vascular congestion, which results in increased gut permeability (Bauer et al., 2001; Gunnarsdottir et al., 2003). Moreover, impaired liver function promotes changes in bacterial communities in the gut through decreased bile acid production and defective intestinal motility that leads to SIBO (Sung, Shaffer, & Costerton, 1993). Thus, it is now well recognized that impaired fluid/liver physiology and innate immunity in combination with dysbiosis are key pathological processes that promote bacterial translocation to the peritoneum. HE is a broad term that encompasses a constellation of neuropsychiatric abnormalities observed in patients with liver dysfunction (Bajaj, 2010). Overt HE is diagnosed in up to 45% of patients with cirrhosis, while minimal HE is observed in 60–80% of the patients (Bajaj, 2010). In healthy individuals, the liver protects the brain from ammonia by converting it to urea, which is then excreted by the kidneys. In the context of severe liver dysfunction, ammonia becomes the critical driver of HE pathogenesis and the intestinal microbiota is by far its predominant source (Williams, 2007). In particular, Urease-producing bacteria such as Klebsiella and Proteus species seem to play a critical role in increased ammonia production and HE development (Basile & Jones, 1997). In concordance with the concept of HE being a bacterial-driven disease, treatment with nonabsorbable antibiotics such as Neomycin and Rifaximinis is associated with a significant decrease in the risk of breakthrough episodes of HE, relapses, or hospitalization due to this neuropsychiatric complication (Bajaj et al., 2011; Bass et al., 2010; Sidhu et al., 2011). Recently, the role of specific bacterial families in cirrhosis has begun to be addressed. Two studies have performed nonculture-based methods to determine the composition of the microbiota in patients with cirrhosis and HE. Both studies found a higher concentration of Streptococcaceae and a negative correlation between cirrhosis and the abundance of Lachnospiraceae (Bajaj et al., 2012; Chen et al., 2011). Interestingly, Bajaj et al. (2012) found that in addition to changes in the intestinal microbiota between healthy and cirrhotic individuals, there was a significant increase in the abundance of different bacterial families (Enterobacteriaceae, Alcaligenaceae, and Streptococcaceae) in patients with confounded HE. Moreover, a positive correlation between cognitive dysfunction and the presence of Alcaligenaceae and Porphyromonadaceae was observed by standardized cognitive testing (Bajaj et al., 2012). The investigation of the gut microbiome in cirrhosis and its correlation to severe clinical

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complications is still in its early stages, but identification of bacterial species that specifically drives disease progression will greatly improve our understanding of the pathogenesis of these complex human diseases.

2.3. Hepatocellular carcinoma HCC is one of the most frequent human cancers worldwide. Approximately 80–90% of HCCs are preceded by chronic liver disease, hepatic fibrosis, and cirrhosis (Nordenstedt, White, & El-Serag, 2010). Therefore, it has been speculated that microbial-derived products are essential determinants of HCC progression. Indeed, recent studies performed using a mouse model of HCC showed that hepatocarcinogenesis in chronically injured livers depended on the intestinal microbiota and Toll-like receptor 4 (TLR4) activation in non-bone-marrow-derived resident liver cells. Importantly, TLR4 and the gut microbiota are not required for HCC initiation but for HCC progression as intestinal sterilization restricted late stages of hepatocarcinogenesis (Dapito et al., 2012). The role of the microbiome on human HCC is an unexplored area that warrants further investigation in the following years.

2.4. Autoimmune liver disease Primary sclerosing cholangitis (PSC) is a chronic liver disease characterized by inflammation and eventual obstruction of biliary ducts (Levy & Lindor, 2006). Although the pathogenesis of PSC remains undetermined, intestinal microbiota is considered to be a major factor in its etiology. The role of intestinal bacterial communities in ulcerative colitis (UC) pathogenesis is well characterized. Interestingly, approximately 75% of patients with PSC have UC and nearly 3% of patients with UC have PSC as a concomitant comorbidity (Bambha et al., 2003; Bergquist et al., 2008; Hashimoto et al., 1993; Joo et al., 2009; O’Toole et al., 2012; Sano et al., 2011; Ye et al., 2011). Moreover, PSC is more frequent in UC patients with total colonic involvement suggesting a strong positive correlation between intestinal inflammation and PSC development (Joo et al., 2009; O’Toole et al., 2012). Several lines of evidence point to the microbiota as a common denominator driving liver and intestinal inflammation in this condition. In the bile of PSC patients, Candida and enteric bacteria such as Escherichia coli are frequently detected (Rudolph et al., 2009). End-stage PSC liver shows

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significantly increased expression and activation of critical genes involved in innate immune pathways (Miyake & Yamamoto, 2013). Finally, serum atypical perinuclear antineutrophil cytoplasmic antibodies (pANCA) are frequently found in patients with PSC (Mulder et al., 1993; Terjung et al., 1998). Recently, the autoantigen of this atypical pANCA has been reported to be b-tubulin, but perhaps more importantly, pANCA crossreacts with FtsZ, a bacterial cytoskeletal protein present in all intestinal bacteria (Terjung et al., 2010). Thus, identifying the specific bacterial species that trigger PCS is a clinically relevant problem that deserves further investigation. Primary biliary cirrhosis (PBC) affects approximately 40 per 100,000 people in the United States. PBC is an autoimmune liver disorder characterized by immune cell activation and directed damage of cholangiocytes, which results in cholestasis that ultimately leads to hepatic fibrogenesis and liver failure in 26% of patients within 10 years of diagnosis (Washington, 2007). The presence in the serum of antimitochondrial antibodies (AMAs) is the hallmark of PBC. AMAs are detected in approximately 95% of PBC patients and their cross-reaction with bacterial components is proposed as a critical event for the early pathogenesis of PBC (Bogdanos et al., 2004; Hopf et al., 1989). AMAs have been reported to react with proteins of E. coli isolated from PBC patients (Bogdanos et al., 2004; Hopf et al., 1989). Moreover, IgG3 antibodies in approximately 50% of PBC patients cross-react with b-galactosidase of Lactobacillus delbrueckii, and in 25% of PBC patients, the serum reacts specifically with proteins of Novosphingobium aromaticivorans from stool specimens (Bogdanos et al., 2005; Selmi et al., 2003). Given this association, further study is warranted to determine if modulation of gut microbiota might aid in the treatment of this catastrophic disease.

3. ROLE OF THE INTERACTIONS BETWEEN THE INNATE IMMUNE SYSTEM AND THE INTESTINAL MICROBIOTA ON CHRONIC LIVER DISEASES The complex interplay between the host and its indigenous microflora is mediated by a large array of pattern-recognition receptors (PRRs) of the innate immune system (Carvalho, Aitken, Vijay-Kumar, & Gewirtz, 2012). Originally mainly appreciated for their role in recognizing invading pathogenic microbes and for the initiation of adaptive immune responses, these receptors and their downstream signaling cascades are increasingly regarded

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as pivotal for the recognition of the commensal microbiota. This microbial recognition plays an important role under homeostatic conditions, and dysfunction in innate signaling in the intestine has been associated with aberrant development of the intestinal immune system, failure in maintenance of intestinal epithelial homeostasis and barrier function, and exacerbated intestinal injury (Michelsen & Arditi, 2007). Importantly, this innate sensing function also serves to locally contain the microbiota and to exclude intestinal microorganisms from the systemic circulation (Slack et al., 2009). The innate receptors expressed in the gastrointestinal tract represent the first line of defense against invasion of microorganisms. However, in cases of increased microbial translocation through the gastrointestinal barrier, the liver as first line of defense requires the expression of innate PRRs in order to set in place a secondary surveillance system of microbial products potentially draining from the gastrointestinal tract. Indeed, intrahepatic expression of innate immune receptors has been described for Kupffer cells (Visvanathan et al., 2007), liver sinusoidal endothelial cells (Hosel et al., 2012), hepatic stellate cells (Wang et al., 2009), biliary epithelial cells (Yokoyama et al., 2006), and hepatocytes (Wang et al., 2005). Consequently, the liver has to master a delicate balance between its ability to induce systemic tolerance toward innocuous food particles and occasional translocation of commensal microbial products and its role in promoting inflammation when a persistent microbial stimulus caused by intestinal breech is indicative of systemic microbial spread. In the following sections, we will discuss how hepatic PRR signaling mediates host–microbial interactions in this vital organ, and how aberrations in PRR expression and signaling contribute to the molecular etiology of liver disease.

3.1. Toll-like receptors TLRs were the first class of PRRs discovered. They recognize a wide range of microbial ligands, ranging from bacterial and fungal cell wall components to nucleic acid (Kawai & Akira, 2010). TLRs are expressed in a wide variety of liver cells and have long been recognized to be involved in the pathogenesis of liver diseases. In particular, Kupffer cells express high levels of TLR2, TLR3, and TLR4, and respond to LPS stimulation with the production of TNF-a, IL-6, and IFN-g. Moreover, the expression of TLRs has been found on hepatocytes, biliary epithelial cells, hepatic stellate cells, and liver sinusoidal endothelial cells (Miyake & Yamamoto, 2013) (Fig. 3.2). The TLR4–MyD88–NF-kB signaling axis has been found to play a critical role in various pathophysiological settings in the liver, including cirrhosis,

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Steatosis

Hepatocyte: TLR2-4

Kupffer cell: TLR2-4

Stellate cell: TLR1-9

TNF IL-6 Inflammatory response in the liver

LSEC: TLR2

Flux of PRR ligands Enterocytes: NLRP6

Intestinal dysbiosis

Figure 3.2 Multiple layers of pattern-recognition receptor involvement in the pathogenesis of liver disease. Functional expression of the NLRP6 inflammasome in the intestine is necessary to avoid dysbiosis. Chronic intestinal inflammation is associated with increased translocation of microbes across the gastrointestinal tract and influx of microbial products into the liver. There, TLR expression on a variety of cell types mediates an aberrant respond to the increased microbial load, initiating an exaggerated inflammatory response that can lead to hepatitis.

fibrosis, viral hepatitis, HCC, and fatty liver disease. For instance, in mice on a high-fat diet, TLR4 deficiency ameliorates hepatic steatosis (Li et al., 2011). In addition, signaling through TRIF downstream of TLR4 in Kupffer cells has been shown to promote alcoholic liver disease (Gao et al., 2011). Further, hepatic TLR4 expression is increased in animal models of NASH (Thuy et al., 2008), PSC (Mueller et al., 2011), and PBC (Wang et al., 2005). These animal studies have been supported by genetic data from humans. A polymorphism in the gene encoding TLR4, which attenuates the signaling downstream of the receptor in response to LPS stimulation, has been associated with a decreased risk to develop cirrhosis (Figueroa et al., 2012; Huang et al., 2007). Another TLR which has been repeatedly associated with enhanced severity of inflammatory liver disease is TLR9, which signals through IRF-7 to induce the expression of type I interferons (IFNs). Interestingly, type I IFNs

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were recently described to protect from TLR9-associated liver damage, and this effect was mediated by the endogenous IL-1 receptor antagonist (Petrasek, Dolganiuc, Csak, Kurt-Jones, & Szabo, 2011). The same authors also found a protective role for type I IFNs in a TLR4-driven model of alcoholic liver disease (Petrasek, Dolganiuc, Csak, Nath, et al., 2011). The involvement of TLRs in a multitude of liver pathologies clearly implied a role for increased microbial translocation across the gastrointestinal tract and hepatic recognition of microbial products (Fig. 3.2), but direct evidence for this notion has been lacking until recently. First insight came from a study by Seki et al. who showed an involvement of the microbiota in the development of hepatic fibrosis. Antibiotic treatment, as well as TLR4- or MyD88-deficiency, reduced fibrosis after bile duct ligation. TLR4 expression on hepatic stellate cells led to enhanced TGF-b signaling and recruitment of Kupffer cells to the fibrotic liver (Seki et al., 2007). As detailed below, we recently described that, under conditions of intestinal inflammation, the influx of microbial products into the liver promotes the development and progression of NAFLD in a TLR4- and TLR9-dependent manner (Henao-Mejia, Elinav, Jin, et al., 2012). In concordance with these results, Lin et al. recently used the concanavalin A (ConA) model of fulminant liver injury to demonstrate that the intestinal microbiota is critically involved in TLR4-mediated hepatitis. Treatment of mice with broad-spectrum antibiotics as well as TLR4 deficiency greatly ameliorated liver damage, as evidenced by reduced release of aminotransferases into the blood, dampened production of proinflammatory cytokines, and decreased hepatic cell death (Lin et al., 2012). In contrast, administration of purified LPS potentiated liver pathology in the ConA model. Adoptive transfer experiments using TLR4-deficient or sufficient splenocytes revealed that immune cells contribute to disease progression through TLR4 expression.

3.2. Inflammasomes Inflammasomes are a group of cytosolic multiprotein complexes, classically consisting of an upstream sensor protein of the NOD-like receptor (NLR) family, the adaptor protein ASC, and the downstream effector caspase-1 (Henao-Mejia, Elinav, Strowig, & Flavell, 2012). To date, the NLR proteins NLRP1, NLRP2, NLRP3, NLRP6, NLRP7, NLRC4, and the HIN-200 family member AIM2 have been reported to initiate the formation of an inflammasome. Upon stimulation with a diverse set of microbial

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or damage-associated molecular patterns, inflammasome assembly leads to the autocatalytic cleavage of caspase-1 and processing of pro-IL-1b and pro-IL-18 into their mature and bioactive forms (Strowig, Henao-Mejia, Elinav, & Flavell, 2012). Inflammasome activity is thought to require two sequential stimuli. The first stimulus drives transcription of the proforms of IL-1b and IL-18, while the second stimulus is required for the formation of the inflammasome complex (Latz, 2010). Inflammasomes fulfill a dual role, recognizing both endogenous damage-associated substances such as ATP or crystal particles and initiating immune responses in reaction to pathogen-associated molecular patterns during bacterial, viral, fungal, and parasitic infections (Elinav, Strowig, Henao-Mejia, & Flavell, 2011). In addition, the inflammasomes are critically involved in the complex interplay between the intestinal immune system and the gut microbiota, which will be covered in more detail below. Recently, inflammasomes were identified to play a role in the pathogenesis of liver disease. Inflammasome components are expressed by various cell types in the liver. Kupffer cells and sinusoidal endothelial cells express high level of NLRP1, NLRP3, and AIM2, and hepatocytes upregulate NLRP3 expression in an LPS-dependent manner (Boaru, Borkham-Kamphorst, Tihaa, Haas, & Weiskirchen, 2012). Imaeda et al. (2009) initially demonstrated an involvement of the NLRP3 inflammasome in the development of acetaminophen-induced hepatotoxicity and showed reduced mortality in acetaminophen-treated mice lacking any component of the NLRP3 inflammasome, although others could not find a role for NLRP3 in acetaminophen-mediated liver failure (Williams, Farhood, & Jaeschke, 2010). Watanabe et al. (2009) revealed expression of inflammasome components in hepatic stellate cells and demonstrated an involvement of the inflammasome in a mouse model of liver fibrosis using carbon tetrachloride or thioacetamide. Similarly, knockdown of NLRP3 ameliorated liver inflammation and protected ischemia–reperfusion injury in mice by preventing excessive production of inflammatory cytokines and NF-kB activity (Zhu et al., 2011). These early studies mainly focused on the role of the inflammasome in the response against tissue damage in sterile injury-mediated models of liver disease. Subsequent reports, however, have also demonstrated an involvement of the inflammasome in liver pathology caused by microbial components or live microorganisms, such as in a model of Propionibacterium acnes-induced sensitization to LPS-induced liver injury (Tsutsui, Imamura, Fujimoto, & Nakanishi, 2010) and in Schistosoma mansoni infection (Ritter et al., 2010).

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In these studies, a cooperative behavior of TLR signaling and inflammasome activation was noticed to be a driving force in the development of overt liver inflammation, suggesting concerted recognition events of microbial- and damage-associated molecules. Interestingly, Csak et al. (2011) recently showed an involvement of the NLRP3 inflammasome in the development and progression of NASH. Upon induction of a mouse model of NASH, expression of inflammasome components was upregulated in the liver and inflammasome activation occurred in isolated hepatocytes. Mechanistically, palmitic acid, a saturated fatty acid, was found to activate the inflammasome and sensitized hepatocytes to IL-1b secretion in response to LPS. The results from this study indicated that both microbial and nonmicrobial PRR ligands act in concert to induce pathogenic inflammasome responses in the liver. A later study confirmed NLRP3 activation in the liver and showed that LPS stimulation alone is sufficient to drive hepatic production of inflammatory cytokines downstream of NLRP3 inflammasome activation (Ganz, Csak, Nath, & Szabo, 2011).

3.3. C-type lectins C-type lectin (CTL) receptors and their downstream adaptor molecules are mediating recognition of glycosylated ligands on microorganisms (Sancho & Reis e Sousa, 2012). Dectin-1 and 2 are two CTLs involved in the immune response against fungal pathogens. The recognition of fungal-associated molecular patterns elicits a downstream cascade through the signaling molecules caspase recruitment domain-containing protein 9 (CARD9) and Syk (Kerrigan & Brown, 2011). A recent study found hepatic mRNA expression in humans of many factors involved in CTL signaling, including Dectin-1, Syk, and CARD9 (Lech et al., 2012). Interestingly, CARD9, which is known as a susceptibility locus in inflammatory bowel disease (IBD), has recently been associated with PSC, along with Rel and IL-2, two other IBD risk loci (Janse et al., 2011). Rel is a member of the NF-kB family of transcription factors, CARD9 induces NF-kB signaling, and IL-2 is an NF-kB target gene, potentially combining all three susceptibility loci into one pathway. The involvement of three members of a fungal recognition pathway in PSC implies a functional role of innate immune recognition of fungal microorganisms in the pathogenesis of this disease. CARD9 is essential for the control of fungal infection, and CARD9-deficient mice show high rates of

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early mortality after infection with Candida albicans (Gross et al., 2006). As mentioned above, Candida is detected in the bile fluid of 1 in every 10 PSC patients. In most cases, the detection of fungi in the bile negatively influences the prognosis on disease severity (Rudolph et al., 2009). Functional studies are needed in the future to delineate the mechanisms and the importance of host–fungal interactions in the pathophysiology of liver disease. Intriguingly, the recently suggested link between alterations in commensal fungal sensing and susceptibility to IBD may potentially provide a mechanistic explanation for the substantial susceptibility for PSC among chronic IBD patients (Iliev et al., 2012). Taken together, the involvement of PRRs of the innate immune system in the pathogenesis of inflammatory liver disease has so far been interpreted in the context of local responses to endogenous signal of damage. While PRR-mediated recognition of damage-associated molecular patterns certainly plays a critical role in disease development and progression, recent evidence indicates that one should also consider microbial ligands as drivers in hepatic inflammatory disorders.

3.4. Dysbiosis associated with innate immune deficiency and its implications for liver disease The cases described above are examples of a liver-intrinsic role of microbial recognition and its association with disease pathogenesis. Recent studies, however, point to a new role of extrahepatic innate immune-microbial cross talk in the initiation and progression of liver disease. First evidence came from a report demonstrating that mice lacking TLR5, the receptor recognizing bacterial flagellin, develop features of metabolic syndrome as a consequence of altered microbial composition in the gut (Vijay-Kumar et al., 2010). Although a recent study has argued that familial transmission, rather than genetic deficiency, might be the dominant driver of dysbiosis in mice (Ubeda et al., 2012), the intriguing notion that defective host–microbiome interactions in the intestine might have consequences that are not limited to regulating inflammation in the gastrointestinal tract, but rather affect systemic metabolism and liver disease, has prompted further investigation. We recently found that the intestinal tracts of mice deficient in the inflammasome components NLRP3, NLRP6, ASC, and Caspase-1, as well as mice lacking the downstream effector cytokine IL-18, harbor an aberrant microbial community which is characterized by the overrepresentation of anaerobic bacterial species of the Prevotellaceae family and the candidate phylum TM7 (Elinav, Strowig, Kau, et al., 2011). This indicates that

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inflammasome activity in the intestine is required for the maintenance of a stable microflora composition, partially through the secretion of IL-18. The altered microbiota found in inflammasome-deficient mice was transferable to wildtype mice upon cohousing in the same cage, demonstrating a dominant population effect, and was reversible upon antibiotic treatment. The biogeographical niche enabling the outgrowth of Prevotellaceae seemed to be the area close to the colonic epithelial layer and the colonic crypts, an area which is normally less densely colonized with microbes due to mechanisms involving antimicrobial peptide production and mucus secretion. This altered intestinal flora leads to mild chronic inflammation and greatly predisposes to experimental colitis. Mechanistically, the colitogenic bacteria present in inflammasome-deficient mice leads to enhanced epithelial production of the chemokine CCL5, which in turn recruits proinflammatory immune cell populations to the intestinal lamina propria (Elinav, Strowig, Kau, et al., 2011). Most importantly, however, we found that the inflammatory processes regulated by the colitogenic flora were not limited to the regulation of local immune responses. When inflammasome-deficient mice were fed a methionine/choline-deficient diet, a model commonly used to induce NAFLD, they featured a dramatic outgrowth of bacterial species of the Porphyromonadaceae family and enhanced translocation of microbial products, in particular, TLR4 and TLR9 ligands, to the portal circulation (HenaoMejia, Elinav, Jin, et al., 2012). Again, this increased microbial translocation across the gastrointestinal tract was dependent on dysbiosis-induced CCL5 production and intestinal inflammation. In the liver, the increased stimulation of TLR4 and TLR9 led to augmented production of TNF-a via MyD88/TRIF signaling, which initiated an inflammatory process leading to the development of NASH. The altered microbiota alone, when transferred from inflammasomedeficient or IL-18-deficient mice to wild-type recipients, was able to enhance susceptibility to NASH in a CCL5-, TLR4-, TLR9-, MyD88/TRIF-, and TNF-dependent manner, demonstrating that dysbiosis, rather than genetic deficiency, was responsible for increased disease susceptibility and that metabolic disease might feature infectious, that is, transmissible microbial, components. Correspondingly, antibiotic treatment of inflammasome-deficient mice fed an MCD diet not only ameliorated NASH severity but also inhibited transmission of the phenotype to wild-type recipients. Moreover, the abnormal microflora also influenced other manifestations of metabolic syndrome in other mouse models of disease. Genetically obese leptin receptor-deficient mice gained markedly more weight when cohoused with inflammasome-deficient mice and so did ASC-deficient

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mice and cohoused wild-type mice fed a high-fat diet. Antibiotic treatment reversed not only weight gain but also fasting plasma insulin amounts and glucose intolerance to normal levels, showing the strong influence of the microbial component on systemic metabolic parameters (Henao-Mejia, Elinav, Jin, et al., 2012). These results demonstrated that homeostatic extrahepatic expression of PRRs is necessary to prevent the development of dysbiosis in the gastrointestinal tract, which in turn predisposes to liver disease via the tight anatomical connection between both organ systems (Fig. 3.2). They also provide an example where multistage host–microbial interactions via different kinds of PRRs and their downstream signaling are involved in disease progression, both at distal (in this case, inflammasomes) and proximal sites (in this case, TLRs). The changes induced by the colitogenic microflora affect inflammatory processes locally (induction of CCL5 and leukocyte recruitment to the intestine), at the most proximal sites draining the intestine (inflammatory cytokine production in the liver), and even beyond (multiorgan regulation of weight gain and insulin sensitivity).

4. PROBIOTICS AND THEIR POTENTIAL ROLE IN LIVER DISEASE THERAPY The recognition of the importance of dysbiosis in the development and progression of liver disease opens new avenues for the development of therapeutic approaches. Similar to diseases in which a contribution of dysbiosis has long been appreciated, such as IBD, therapeutic intervention with the aim of adjusting the composition of the intestinal microflora might prove a valuable tool in the treatment of liver diseases. Probiotics and prebiotics are actively exploited for their therapeutic effects in IBD (DuPont & DuPont, 2011). Probiotics are live microorganisms given as dietary supplements to modify their relative representation in the intestinal ecosystem, while prebiotics are nondigestible dietary substances which promote the growth of one or more types of microorganisms in the gut, with the aim of increasing their relative abundance in the intestinal microflora. The benefits of pro- and prebiotics include direct effects, such as the increased release of metabolic products, and indirect effects, in particular, through microbe–microbe interactions and changes in population dynamics of intestinal microbial communities. Interestingly, the initial studies have demonstrated beneficial effects of probiotic interventions in liver disease. In a rat model of liver damage

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provoked by ischemia–reperfusion, intestinal dysbiosis was observed, including the outgrowth of Enterobacteriaceae and a decrease in Bacteroides spp., Lactobacillus spp., and Bifidobacter spp. These changes could be reversed by dietary supplementation with Lactobacillus paracasei, which remarkably led to reduced liver inflammation, as evidenced by ameliorated production of the proinflammatory cytokines IL-1b, IL-6, and TNF-a (Nardone et al., 2010). Similarly, after chemical liver injury, probiotic therapy with Lactobacillus spp. reduced hepatic inflammation by supporting intestinal barrier function and reducing microbial translocation across the gastrointestinal tract (Osman, Adawi, Ahrne, Jeppsson, & Molin, 2007). Interestingly, in our inflammasome dysbiosis mouse model, representation of Lactobacillus was significantly reduced (Elinav, Strowig, Kau, et al., 2011), pointing toward potential involvement of this commensal family in prevention of local mucosal inflammation and the related tendency toward systemic metabolic complications. Indeed, probiotic interventions were shown to influence hepatic metabolism, as was demonstrated in a rat model of high-cholesterol diet, in which Lactobacillus spp. supplementation in the food reduced the levels of cholesterol and triglycerides in the liver (Xie et al., 2011). Further, HE in cirrhotic patients was ameliorated by probiotic therapy leading to decreased representation of E. coli and reduced blood ammonia levels (Liu et al., 2004). Future studies are clearly needed to understand the mechanisms by which dietary manipulation of the intestinal ecosystem exerts its effects on liver metabolism. Gnotobiotic mice represent an excellent tool to study the contribution of individual microorganisms and their metabolic pathways to liver function.

5. CONCLUSIONS The mesenteric lymph node is the “first pass” organ for nutrients and microbial substances entering the lymph fluid in the intestinal lamina propria. As such, it serves as a key site for tolerance induction to food particles but at the same time acts as a firewall to prevent systemic spread of microorganisms. Similarly, the liver is exposed to all substances leaving the gastrointestinal tract via the portal blood circulation and faces similar challenges balancing tolerance to innocuous particles draining from the intestine and barrier function to potentially harmful microbial substances. In contrast to the mesenteric lymph node, the liver is the body’s prime metabolic organ, and any aberrations from the homeostatic state of host–microbial interactions in the liver may affect its metabolic functions.

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We are convinced that the realization that both intrahepatic and extrahepatic host–microbial interactions, and in particular, innate immune system–microflora interactions, drastically influence systemic physiologic and pathophysiologic processes will guide future efforts to exploit this new insight in preclinical and clinical settings.

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