Gut microbiota, fatty liver disease, and hepatocellular

Liver Research xxx (2018) 1e9

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Liver Research journal homepage: http://www.keaipublishing.com/en/journals/liver-research

Review Article

Gut microbiota, fatty liver disease, and hepatocellular carcinoma* Huikuan Chu a, b, Brandon Williams b, Bernd Schnabl b, c, * a

Division of Gastroenterology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Department of Medicine, University of California San Diego, La Jolla, CA, USA c Department of Medicine, VA San Diego Healthcare System, San Diego, CA, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 August 2017 Received in revised form 31 October 2017 Accepted 7 November 2017

Intestinal bacteria contribute to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Recently developed microbial profiling techniques are beginning to shed light on the nature of the changes in the gut microbiota that accompany NAFLD and non-alcoholic steatohepatitis (NASH). In this review, we summarize the role of gut microbiota in the development of NAFLD, NASH, and hepatocellular carcinoma (HCC). We highlight the mechanisms by which gut microbiota contribute to NAFLD/NASH, including through alterations in gut epithelial permeability, choline metabolism, endogenous alcohol production, release of inflammatory cytokines, regulation of hepatic Toll-like receptor (TLR), and bile acid metabolism. In addition, we analyze possible mechanisms for enhanced hepatic carcinogenesis, including alterations in bile acid metabolism, release of inflammatory cytokines, and expression of TLR-4. Finally, we describe therapeutic approaches for NAFLD/NASH and preventive strategies for HCC involving modulation of the intestinal microbiota or affected host pathways. Although recent studies have provided useful information, large-scale prospective studies are required to better characterize the intestinal microbiota and metabolome, in order to demonstrate a causative role for changes in the gut microbiota in the etiology of NAFLD/NASH, to identify new therapeutic strategies for NAFLD/NASH, and to develop more effective methods of preventing HCC. © 2018 The Third Affiliated Hospital of Sun Yat-sen University. Publishing Services by Elsevier B. V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: Gut microbiota Intestinal microbiome Metabolome Metagenome Fatty liver disease Non-alcoholic fatty liver disease (NAFLD) Non-alcoholic steatohepatitis (NASH) Hepatocellular carcinoma (HCC)

1. Introduction Non-alcoholic fatty liver disease (NAFLD) is currently the most common liver disease worldwide in both adults and children.1e3 NAFLD is defined as the presence of hepatic steatosis in the absence of any secondary causes of hepatic fat accumulation, including excessive alcohol consumption, hepatitis C (genotype 3), Wilson's disease, lipodystrophy, starvation, parenteral nutrition, abetalipoproteinemia, medication (e.g., amiodarone, methotrexate, tamoxifen, corticosteroids, valproate, or antiretroviral medications), Reye's syndrome, acute fatty liver of pregnancy, HELLP (hemolysis, elevated liver enzymes, and low platelet count) syndrome, and inborn errors of metabolism (e.g., lecithin cholesterol acyltransferase deficiency, cholesterol ester storage disease, or Wolman disease).2

*

Edited by Peiling Zhu and Genshu Wang. * Corresponding author. Department of Medicine, University of California San Diego, Biomedical Research Facility 2 (BRF2), La Jolla, CA, USA. E-mail address: [email protected] (B. Schnabl).

NAFLD includes a spectrum of pathologies, from simple steatosis, defined as the presence of steatosis without any histologic or biochemical injury, to non-alcoholic steatohepatitis (NASH), characterized by steatosis, inflammation, and hepatocyte injury (ballooning), with or without cirrhosis. Patients with NASH have a high likelihood of developing advanced fibrosis and cirrhosis. It is estimated that about one third of the cases of early-stage NASH will progress to stage 3 or 4 fibrosis (cirrhosis) over 5e10 years.4 Additionally, patients with NAFLD are at a higher risk of developing hepatocellular carcinoma (HCC), even in the absence of cirrhosis. In Western countries, 4%e22% of HCC cases are now attributed to NAFLD and the risk of developing HCC for patients with NASH and cryptogenic cirrhosis is between 2.4% over 7 years and 12.8% over 3 years.5e7 NAFLD pathophysiology is multifactorial, involving ecological, genetic, and metabolic factors, such as high energy intake, limited physical activity, and a poorly balanced diet.8,9 Together with epigenetic factors, these promote insulin resistance and hepatic fat accumulation.8,10 The development and progression of inflammation in the steatotic liver was initially proposed to be due to endotoxemia resulted from greater gut

https://doi.org/10.1016/j.livres.2017.11.005 2542-5684/© 2018 The Third Affiliated Hospital of Sun Yat-sen University. Publishing Services by Elsevier B. V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: Chu H, et al., Gut microbiota, fatty liver disease, and hepatocellular carcinoma, Liver Research (2018), https:// doi.org/10.1016/j.livres.2017.11.005

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H. Chu et al. / Liver Research xxx (2018) 1e9

permeability.11,12 Recently, however, studies have reported that the intestinal microbiota plays an important role in the pathogenesis of NAFLD.13e15 In addition, obesity induces changes in the composition of the gut microbiota and its metabolites that promote the development of HCC.16 The development of microbial profiling techniques has shed new light on such changes in the gut microbiota and the role of gut bacteria in the development of NAFLD.17 In this review, we focus on the mechanisms by which gut microbiota affect the development of NAFLD/NASH and their progression to HCC. Finally, we discuss potentially effective methods to treat NAFLD/NASH and prevent the development of HCC by modulating the intestinal microbiome.

intestinal fluid. Various breath tests, including the glucose-H2 breath test have been used to quantify SIBO, given the difficulty in obtaining cultures.24 Patients with SIBO have a greater risk of hepatic steatosis,25 and SIBO and the presence of metabolic syndrome were independently associated with more severe hepatic steatosis.24 SIBO has an important role in NASH development and progression, because it increases intestinal permeability by disrupting intercellular tight junctions in the gut, enhances hepatic expression of Toll-like receptor (TLR)-4, and leads to release of interleukin (IL)-8.22,23,26 4. Mechanisms by which the intestinal microbiota impacts NAFLD/NASH

2. Gut dysbiosis is associated with NAFLD/NASH and HCC 4.1. Gut barrier dysfunction Intestinal dysbiosis is associated with a variety of liver diseases, including NAFLD and NASH.18 However, there have been few studies to date on the relationship between the gut microbiome and NAFLD in humans, and these have yielded inconsistent results (Table 1). For example, patients with NASH had significantly lower levels of Bacteroidetes compared to subjects with simple steatosis and healthy individuals,13 and another study generated similar results in children,19 whereas a third study reported larger numbers of Prevotella, one of the most important genera within the phylum Bacteroidetes, in NASH patients compared to healthy subjects.15 Selected members of the phylum Firmicutes were also more frequent in NAFLD than in non-obese controls (healthy people),14 but lower numbers of Firmicutes were present in NASH patients in another study.15 Finally, while one study reported larger numbers of the genus Escherichia from the Enterobacteriaceae family in subjects with NASH,15 there was no significant difference in Escherichia coli numbers between NASH patients and subjects with simple steatosis in another paper.13 Taken together, these studies suggest that the composition of the gut microbiota does differ in patients with NAFLD/NASH and controls, but the specific data obtained to date have been inconsistent. Further studies have also investigated the association between gut dysbiosis, severe NAFLD, and HCC (Table 2). Numbers of the genera Bacteroides, Prevotella, and Ruminococcus significantly differ between patients with F0 or F1 fibrosis and those with significant (F 2) fibrosis (according to the NASH Clinical Research Network scoring system, F0 ¼ no fibrosis, F1 ¼ perisinusoidal or portal/periportal fibrosis, F2 ¼ perisinusoidal and portal/periportal fibrosis, F3 ¼ bridging fibrosis, and F4 ¼ cirrhosis). The abundance of Bacteroides and Ruminococcus is significantly greater in F 2 patients, whereas Prevotella is less abundant.20 Firmicutes are more abundant in mild/moderate NAFLD, while Proteobacteria is more common in advanced fibrosis. At the species level, Eubacterium rectale and Bacteroides vulgatus are the most abundant organisms in mild/ moderate NAFLD, while B. vulgatus and E. coli are the most abundant in advanced fibrosis. Finally, Ruminococcus obeum CAG: 39, R. obeum, and E. rectale are significantly less abundant in advanced fibrosis than in mild/moderate NAFLD.21 Thus, NAFLD severity is associated with gut dysbiosis. In addition, alterations in the gut microbiota induced by obesity could promote the development of HCC.16 3. NAFLD/NASH is associated with bacterial overgrowth in the small intestine Patients with NAFLD/NASH not only have compositional changes in the gut microbiota but also likely to have small intestinal bacterial overgrowth (SIBO), with an estimated prevalence ranging from 50% to 77.78%.12,22,23 SIBO is defined as a total bacteria growth of more than 105 colony-forming units per milliliter (CFU/mL) of

The intestinal microbiota plays a critical role in the maintenance of the integrity of the intestinal barrier,27 and changes in intestinal epithelial permeability are associated with the development of NAFLD/NASH.28 Mice with defects in intestinal barrier function develop more severe steatohepatitis than control mice fed a diet high in saturated fat, fructose, and cholesterol (HFCD). The colon of patients with NAFLD has lower levels of junctional adhesion molecule A (JAM-A) and higher levels of inflammation than subjects without NAFLD.28 The gut immune system is altered during high fat diet (HFD) feeding.29 In these studies, intestinal permeability was determined by measuring urinary excretion of 51Crethylenediaminetetra-acetate and evaluating tight junctions in duodenal biopsies by immunohistochemical analysis of the zona occludens. Studies of animal models have shown correlations between hepatic inflammation and dysfunction of the intestinal mucosal barrier, which suggest that intestinal mucosal barrier malfunction influences the pathogenesis of NAFLD and possibly NASH.30,31 In addition, patients with NAFLD had a high prevalence of SIBO, indicating that alterations in the microbiota may have contributed to the disruption of gut barrier integrity.22 Epithelial microvillus atrophy, disrupted tight junctions, and SIBO were present in patients eating a HFD, and these changes were more severe in those with NASH than in those with NAFLD.32 Administration of oral antibiotics or sequestration of bacterial endotoxins with sevelamer hydrochloride reduced mucosal inflammation and restored normal liver histology in F11r/ (the F11r gene encodes JAM-A) mice fed a HFCD,28 while treatment of HFD-fed mice with the local gut antiinflammatory drug, 5-aminosalicylic acid (5-ASA), reduced gut inflammation, improved intestinal barrier function, improved metabolic parameters, and reduced hepatic steatosis.29 Altered intestinal barrier function may be involved in the pathogenesis of NAFLD/NASH by exposing the liver to higher levels of bacterial inflammatory products, such as lipopolysaccharide (LPS), and to the toxic bacterial metabolites, which include ethanol, and other volatile organic compounds (VOCs), such as acetone and butanoic acid. 4.2. Choline metabolism Choline is an important constituent of cell and mitochondrial membranes. Acetylcholine plays critical roles in diverse physiological processes in the liver, including lipid metabolism, signaling through lipid second messengers, enterohepatic circulation of bile acids, and cholesterol metabolism.33 Choline is an essential nutrient and low-choline diets can cause health problems in humans, including fatty liver disease.34,35 Choline levels are not only influenced by dietary and genetic factors,36 but are also modulated by the gut microbiota.37,38 Subjects consuming a low



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Table 1 Changes in the intestinal microbiota associated with NAFLD/NASH. References Diseases and groups

Comparison Implicated microbiota

Mouzaki et al.13

Healthy vs. BacteroidetesY NASH Steatosis vs. Bacteroidetes Y NASH Firmicutes

Healthy (n ¼ 17), Steatosis (n ¼ 11), NASH (n ¼ 22)

Del Children: Chierico Healthy 19 et al. (n ¼ 54), NAFLD (n ¼ 27), NASH (n ¼ 26), Obese (n ¼ 8)

Zhu et al.15 Children: Healthy (n ¼ 16), Obese (n ¼ 25), NASH (n ¼ 22)

Phylum

Healthy (n ¼ 30), NAFLD (n ¼ 30)

Methodology Order

Family

Genus Quantitative real-time PCR (Stool sample)

Lachnospiraceae

Healthy vs. BacteroidetesY all patients

RikenellaceaeY Porphyromonadaceae Bacteroidaceae Ruminococcaceae Lachnospiraceae

Firmicutes[

Firmicutes[ BacteroidetesY ProteobacteriaY ActinobacteriaY NAFLD vs. ProteobacteriaY Obese ActinobacteriaY Healthy vs. Bacteroidetes NASH

Clostridium coccoides [

ParabacteroidesY Bacteroides fragilisY OscillospiraY Dorea[, Ruminococcus[

16S rRNA gene pyrosequencing (Stool sample)

NAFLD vs. NASH

BacteroidaceaeY

BacteroidesY

Healthy vs. Bacteroidetes [ Obese Firmicutes Y

Prevotellaceae [ Rikenellaceae Y Lachnospiraceae Y

Healthy vs. Bacteroidetes [ NASH Firmicutes Y

Eubacteriaceae Ruminococcaceae Y Prevotellaceae [ Rikenellaceae Y Lachnospiraceae Y

Prevotella [ Alistipes Y Blautia Y, Coprococcus Y, RoseburiaY Eubacterium Y

Obese vs. NASH Raman et al.14

Class

Actinobacteria Y Proteobacteria [ Proteobacteria [

Healthy vs. Firmicutes NAFLD

Proteobacteria Bacteroidetes

Eubacteriaceae Ruminococcaceae Y Bifidobacteriaceae Y


Prevotella [ Alistipes Y Blautia Y, Coprococcus Y Eubacterium Y Oscillospira Y Bifidobacterium Y

Gammaproteobacteria Enterobacteriales Enterobacteriaceae [ Escherichia [ Gammaproteobacteria Enterobacteriales Enterobacteriaceae[ Bacilli Clostridia

Lactobacillales Clostridiales

Lactobacillaceae [ Lachnospiraceae [

Clostridia Clostridia Alphaproteobacteria Gammaproteobacteria Bacteroidia

Clostridiales Clostridiales Kiloniellales Pasteurellales Bacteroidales

Ruminococcaceae Y Veillonellaceae[ Kiloniellaceae[ Pasteurellaceae[ Porphyromon adaceaeY

Escherichia [ Lactobacillus [ Robinsoniella [, Roseburia [, Dorea[ Oscillibacter Y


Comparison of condition A vs. condition B: [ signifies an increase in condition B relative to condition A. Y signifies a decrease in condition B relative to condition A. Abbreviations: NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis.

choline diet had variable populations of Gammaproteobacteria, but they vanished from most of the subjects when high dietary choline levels were restored, suggesting that the level of dietary choline influences the composition of the gut microbiome.39 Conversely, the gut microbiome can also affect choline levels. Enzymes produced by the gut microbiota convert dietary choline to dimethylamine (DMA) and trimethylamine (TMA),40,41 which are absorbed through the microvilli and transported via the portal vein to the liver, where they may promote liver inflammation. Furthermore, low choline levels have been associated with fatty liver.39,42e44 Germ-free mice do not excrete TMA, providing solid evidence for the critical role of gut microbiota in the conversion of choline to TMA.45 Exposure to TMA increases the pH of the growth medium of exposed bacteria, resulting in modifications to antibiotic uptake and transient alterations in antibiotic resistance,

providing an alternative means whereby choline and its metabolites can affect community behavior and structure in physically separated bacteria.46 Thus, the gut microbiome may affect the pathogenesis of fatty liver disease by modifying choline metabolism. However, future studies should assess to what extent the changes in the composition of the microbiota described in NAFLD affect choline metabolism and investigate novel therapeutic strategies to regulate choline metabolism. 4.3. Endogenous ethanol production Ethanol is an important microbial metabolite, and it is principally metabolized to acetate and acetaldehyde. Ethanol can inhibit the tricarboxylic acid cycle and increase production of acetate, which is a substrate for fatty acid synthesis,47 while acetaldehyde


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Table 2 Changes in the intestinal microbiota associated with the severity of NAFLD. References

Diseases and groups

Comparison

Implicated microbiota Phylum

Boursier et al.20

Loomba et al.21

F0/1 fibrosis without NASH (n ¼ 20), F0/1 fibrosis with NASH (n ¼ 10), fibrosis F 2 (n ¼ 27)

No NASH vs. NASH F0/1 fibrosis vs. F 2 fibrosis

Mild/moderate (Stage 0e2 fibrosis) NAFLD (n ¼ 72), Advanced fibrosis (Stage 3 or 4 fibrosis) (n ¼ 14)

Stage 0e2 fibrosis Proteobacteria[ Firmicutes Y vs. stage 3 or 4 fibrosis

Methodology

Class Order Family

Bacteroidetes Bacteroidetes Firmicutes

Genus/species

Bacteroidaceae[ PrevotellaceaeY Bacteroidaceae[ PrevotellaceaeY Ruminococcaceae ErysipelotrichaceaeY

Bacteroides[ PrevotellaY Bacteroides[ PrevotellaY Ruminococcus[

Eubacteriaceae Ruminococcaceae

Eubacterium rectaleY Ruminococcus obeum CAG:39Y, Ruminococcus obeumY

16S r RNA genepyro sequencing (Stool sample) Whole genome shotgun sequencing of DNA (Stool sample)

Comparison of condition A vs. condition B: [ signifies an increase in condition B relative to condition A. Y signifies a decrease in condition B relative to condition A. Abbreviations: NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis.

has distinct oxidation-dependent metabolic and cytotoxic effects that can result in liver injury.48 Serum alcohol concentration is high in adult NAFLD patients, and pediatric NASH patients exhibit higher serum alcohol concentrations than healthy controls and non-NASH obese patients.15,49,50 Breath and plasma ethanol levels were also significantly higher in ob/ob mice than in lean control mice.51 In one study there was a significant increase in the abundance of Escherichia in NASH patients.15 Escherichia is a genus of the Enterobacteriaceae family, which may increase the production of ethanol using a mixed-acid fermentation pathway.52e55 In addition, insulin resistance followed by a decrease in alcohol dehydrogenase activity in the liver may alter ethanol metabolism.50 Interestingly, ethanol is also produced by intestinal microbiota cultured under special nutritional conditions.56 The endogenous production of ethanol might, in turn, contribute to the formation of free fatty acids and oxidative stress, further underscoring the potential role of ethanol-producing bacteria in the pathogenesis of NAFLD. Further studies are required to determine the effects of endogenous production of ethanol on the progression of NAFLD and NASH. 4.4. Dysregulation of inflammatory cytokine release Inflammatory cytokines are thought to play a key role in NAFLD/ NASH. Serum tumor necrosis factor alpha (TNF-a) levels are high in patients with NASH,57 while expression of TNF-a and its p55 receptor in the liver of patients with NASH is higher than in patients with simple steatosis.58 Furthermore, a TNF-a gene promoter polymorphism at position 238 may be associated with an increased risk of NAFLD,59 and SIBO in NASH patients is associated with high circulating TNF-a levels.12 Inflammasome deficiency-associated changes in the Firmicutes and Bacteroidetes populations are associated with worsening of hepatic steatosis and inflammation via TLR-4 and TLR-9 activation, which leads to higher hepatic TNF-a expression and thus progression of NASH.60 Conversely, probiotic therapy significantly reduces TNF-a, steatosis, and the NASH activity index.61,62 Thus, the gut microbiota may play a role in the pathogenesis of NASH by regulating TNF-a levels. Another inflammatory cytokine, IL-8, is also involved in NASH.57 The A/A genotype of the IL-8 gene is associated with the progression of NASH,63 while the high prevalence of SIBO in NASH patients is associated with greater expression of TLR-4 and release of IL-8.23 IL18 also has an effect, slowing the progression of NAFLD/NASH by regulating the gut microbiota.60 Further studies are required to explore potential novel therapeutic strategies targeting inflammatory cytokines for patients with NAFLD/NASH.

4.5. Regulation of hepatic TLRs TLRs are pattern recognition receptors that play an important role in host defense by recognizing pathogen-associated molecular patterns (PAMPs). TLR-4 is a receptor for LPS, which is a component of gram-negative bacteria; TLR-9 recognizes DNA containing unmethylated CpG motifs, which are common in bacterial DNA;64,65 TLR-2 binds components of gram-positive bacterial cell walls, such as peptidoglycan and lipoteichoic acid;64,65 and TLR-5 recognizes flagellin, the major protein constituent of bacterial flagella.66,67 Recent studies indicate that TLR signaling plays an important role in the progression of NAFLD and NASH,68e70 and conversely the gut microbiota regulates the expression of TLR in patients with NAFLD/NASH. NASH patients also have a higher prevalence of SIBO, which is associated with enhanced expression of TLR-4 in the liver.23 Inflammasome deficiency-associated changes in the gut microbiota lead to worsening of hepatic steatosis and inflammation through activation of TLR-4 and TLR-9, and thus greater hepatic TNF-a expression and NASH progression.60 The TLR-9 signaling pathway induces production of IL-1b by Kupffer cells, leading to steatosis, inflammation, and fibrosis.71 Mice genetically deficient in TLR-5 exhibit hyperphagia and develop features of the metabolic syndrome, which correlate with changes in the composition of the gut microbiota. In addition, transfer of microbiota from TLR-5 knockout mice to wild-type germ-free mice confers many features of the metabolic syndrome on the recipients.72 These findings suggest that interactions between TLR-5 and specific gut microbiota contribute to the development of the metabolic syndrome. Interestingly, probiotic treatment increased anti-inflammatory cytokine secretion in a TLR-2-dependent manner,73 while Clostridium butyricum induced IL-10 production from intestinal macrophages in acute experimental colitis through TLR-2,74 which suggests that TLR-2 has a dual function. TLR-2 ligands from probiotic bacteria are anti-inflammatory, whereas TLR-2 ligands from the bacteria that are present during obesity induce inflammation. In summary, the gut microbiota may be involved in the pathogenesis of NAFLD and NASH by regulating TLRs, and further studies are needed to identify potential novel therapeutic strategies targeting TLRs for patients with NAFLD/NASH. 4.6. Alterations in bile acid metabolism Bile acids and their metabolites play important roles in maintaining hepatic glucose, cholesterol, and triglyceride homeostasis,



and both bile acid homeostasis and the associated signaling pathways are dysregulated in NAFLD.75 The concentrations of serum bile acids, especially the taurine-conjugated and glycine-conjugated primary and secondary bile acids, are higher in patients with NASH than in healthy volunteers.76 In addition, the gut microbiota has a significant impact on bile acid metabolism,77 and bile acids can have direct effects on the intestinal microbiota by disrupting membranes.78 Thus, there appears to be an association between the gut microbiota and bile acid metabolism. HFD-induced changes in the bile acid pool are mediated through altered gut microbiota,79,80 which in turn contributes to the occurrence of NAFLD by influencing lipid and energy metabolism. Bile acids may also influence NAFLD progression through regulation of the farnesoid X nuclear receptor (FXR) and the G-proteincoupled receptor (GPCR) TGR5.75 Bile acids may have a role in the regulation of insulin sensitivity by activating TGR5,81 and indeed the treatment of diet-induced obese mice with the TGR5 agonist INT-777 leads to reduced body weight gain and increased energy expenditure in brown adipose tissue.82 The gut microbiota may also affect bile acid metabolism by activating FXR during the onset and progression of hepatic steatosis.83e86 Evidence for this comes from observation of the effects of the gut-restricted FXR agonist fexaramine (Fex), which can reduce diet-induced weight gain, systemic inflammation, and hepatic glucose production, while enhancing thermogenesis and browning of white adipose tissue, thereby reducing obesity and insulin resistance.87 Furthermore, 6ethylchenodeoxycholic acid (obeticholic acid) is a potent activator of FXR that ameliorates histological defects in NASH patients.88 However, FXR is able to serve either as an activator or as a direct repressor when it forms a heterodimer with retinoid X receptor a (RXRa/NR2B1) or as a monomer.75 Animals with intestine-specific Fxr disruption had lower hepatic triglyceride accumulation in response to a HFD,84 but both bile acid receptors (FXR and TGR5) may represent important targets for the treatment of the metabolic syndrome,89,90 because administration of TGR5/FXR agonists in these mice improved NAFLD histology and reduced hepatic inflammation by inhibiting the production of inflammatory cytokines by macrophages.91 Indeed, FXR/TGR5 activation regulates the immune phenotype of monocytes and macrophages, both in vitro and in vivo, identifying them as potential targets for the treatment of NAFLD. Taken together, the role of the complex interplay between the microbiota and human bile acids requires further elucidation with respect to the pathogenesis of NAFLD and NASH.

4.7. Other factors that contribute to the pathogenesis of NAFLD/ NASH Enteric bacteria decrease the expression of fasting-induced adipocyte factor (Fiaf) in the gut, thereby inducing lipoprotein lipase (LPL) activity and thus greater hepatic accumulation of triglycerides,92 implying a direct link between the intestinal microbiota and fat deposition in the liver. In addition, gut microbiota uses the host's diet to generate energy, a process which involves the production of SCFAs, including acetate, propionate, and butyrate. The presence of additional SCFAs can increase the quantity of energy-producing substrates absorbed from the intestinal tract, thus contributing to the development of obesity and obesityrelated metabolic diseases.93,94 The dysbiosis-associated factors contributing to NAFLD and NASH are illustrated in Fig. 1.

5

5. How does the intestinal microbiota contribute to the development of cirrhosis and HCC in NAFLD? NAFLD has the potential to evolve into cirrhosis and HCC and the gut microbiota plays an important role in the pathogenesis of cirrhosis and HCC.95 Mice kept under germ-free conditions develop fewer and smaller HCCs than mice housed conventionally, while chronic treatment with a low, non-toxic dose of LPS leads to a significant increase in the number and size of HCCs.96 Furthermore, Yoshimoto et al.16 showed that the administration of antibiotics and gut sterilization could reduce the prevalence of HCCs in obese mice. What is the mechanism by which the gut microbiota contributes to NAFLD-associated cirrhosis and HCC? Firstly, dysbiosis of the gut microbiota leads to an increase in secretion of inflammatory cytokines, such as TNF-a and IL-8, which play a key role in the induction and progression of NAFLD to NASH and cirrhosis.97 Consistent with this, serum TNF-a and IL-8 levels were significantly higher in patients with NASH and cirrhosis.57 Secondly, intestinal dysbiosis leads to activation of TLR-4 and TLR-9, resulting in production of IL1b by Kupffer cells. IL-1b promotes lipid accumulation and cell death in hepatocytes, causing steatosis and inflammation, and stimulates hepatic stellate cells (HSCs) to produce fibrogenic mediators, which promote fibrosis.68,71 In addition, TLR-4 might be directly involved in the pathogenesis of HCC.96 Thirdly, dysbiosis could promote the development of NAFLD-associated HCC by modifying bile acid metabolism. Alterations in the composition of the gut microbiota can result in higher levels of deoxycholic acid (DCA), which provokes a senescence-associated secretory phenotype (SASP) in hepatic stellate cells (HSCs), which in turn secrete various inflammatory and tumor-promoting factors in the liver, thus promoting the development of HCCs.16 In summary, the intestinal microbiota may promote the development of NAFLDassociated cirrhosis and HCC by increasing inflammatory cytokine secretion, activating TLR-4 and TLR-9, and modifying bile acid metabolism (Fig. 2). However, further studies are required to investigate the complex interplay between the microbiota and NAFLD-associated cirrhosis and HCC.

6. Modulation of the intestinal microbiota and treatment of NAFLD/NASH Recent studies of the role of the gut microbiota in the development of NAFLD have provided important evidence to guide the development of a gut microbiota-targeted strategy to prevent or treat NAFLD/NASH and HCC. The most commonly used means of manipulating gut microbiota include the use of probiotic, prebiotic, or synbiotic supplements, or antibiotic treatment. In particular, Lactobacillus and Bifidobacterium are frequently used for their beneficial effects on NAFLD. Probiotic therapies may ameliorate insulin resistance by reducing serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), gammaglutamyltransferase (GGT), total cholesterol, and TNF-a in NAFLD patients.98,99 Probiotic treatment has been shown to reduce intrahepatic triglyceride content, AST, TNF-a, C-reactive protein (CRP), homeostasis model assessment of insulin resistance (HOMA-IR), serum endotoxin, steatosis, and the NASH activity index in patients with NASH.61,100 Probiotics can also reduce the levels of ALT, AST, cholesterol, low-density lipoprotein-C (LDL-C), triglycerides, and waist circumference in children with NAFLD.101 In animal models, probiotics protect against the onset of fructose-induced NAFLD by reducing expression of the tight junction protein occludin in the


6


Fig. 1. Effects of the intestinal microbiota on NAFLD/NASH. HFD feeding results in dysbiosis and intestinal bacterial overgrowth. Dysbiosis leads to greater production of ethanol (EtOH) and SCFA, which are metabolized in the liver. Dietary choline is metabolized by the intestinal microbiota to form TMA, resulting in choline deficiency and hepatic steatosis. The intestinal microbiota suppresses expression of the Fiaf gene by intestinal epithelial cells, resulting in enhanced activity of LPL and greater liberation of free fatty acids (FFA). Greater intestinal permeability leads to translocation of microbial products to the liver and causes inflammation through activation of TLRs and production of inflammatory cytokines, which can drive NAFLD/NASH progression. Dysbiosis also affects bile acid metabolism, which influences the progression of NAFLD through modulation of FXR and TGR5. Abbreviations: HFD, high fat diet; SCFA, short-chain fatty acid; TMA, trimethylamine; Fiaf, fasting-induced adipocyte factor; LPL, lipoprotein lipase; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; FXR, farnesoid X nuclear receptor; TGR5, G-protein-coupled receptor; TLR, toll-like receptor.

Fig. 2. Effects of the intestinal microbiota on HCC tumorigenesis. Long term ingestion of a HFD results in dysbiosis and intestinal bacterial overgrowth. Increased intestinal permeability leads to translocation of microbial products to the liver and causes inflammation and TLR activation, which can stimulate hepatic stellate cells (HSCs) to produce profibrotic factors. In addition, changes in the gut microbiota can result in higher levels of DCA, which provoke a senescence-associated secretory phenotype (SASP) in HSCs. All of these factors promote the development of HCCs. Abbreviations: HCC, hepatocellular carcinoma; HFD, high fat diet; TLR, Toll-like recepter; HSCs, hepatic stellate cells; DCA, deoxycholic acid; SCFA, short-chain fatty acid.

duodenum, attenuating activation of the TLR-4 signaling cascade, and increasing peroxisome proliferator-activated receptor gamma (PPAR-g) activity.102 Probiotics also limit oxidative and inflammatory liver damage in patients with NAFLD by increasing PPAR-a activity and reducing TNF-a levels, metalloproteinase 2 and metalloproteinase 9 activities, and expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2.103 In particular, Lactobacillus casei strain Shirota (LcS) administration markedly suppressed the development of methionine and choline-deficient diet-induced NASH, lowered serum LPS concentrations,

suppressed inflammation and fibrosis in the liver, and reduced inflammation of the colon,104 while administration of Bifidobacteria attenuated NAFLD by modulating the sirtuin1-mediated signaling pathway.105 In contrast, Lactobacillus rhamnosus GG (LGG) reduced duodenal nuclear factor-kB inhibitor (IkB) protein expression and restored portal LPS and duodenal tight junction protein concentrations, while also tending to attenuate TNF-a, IL-8R, and IL-1b mRNA expression in the liver.106 In addition, LGG decreased cholesterol levels, mediated by suppression of FXR and fibroblast growth factor 15 signaling.107



Serum AST was significantly lower in NASH patients treated with a combination of Bifidobacterium (Bifidobacterium longum) and fructooligosaccharide than in the control group,61 which implies a potential benefit of the use of synbiotics for the treatment of NASH. Furthermore, Busset al.108 performed a systematic review of three randomized controlled trials that had tested the efficacy of probiotics, prebiotics or both (synbiotics) for the treatment of NAFLD in adult patients, and showed reductions in aminotransferases in the probiotic- and synbiotic-treated groups. Prebiotic fiber supplements have a beneficial effect in NAFLD because they modify the composition of the gut microbiota, and hence reduce body fat and improve glucose metabolism.109 Therefore, prebiotics, probiotics or both (synbiotics) may hold promise for the treatment of patients with NAFLD in clinical practice. Transplantation with better defined microbial communities or engineered bacterial strains, and substances targeting inflammatory cytokines, choline metabolism, hepatic TLR regulation, and bile acid metabolism might provide novel therapeutic strategies. 7. Conclusions The pathophysiology of NAFLD involves many factors, and the intestinal microbiota plays an important role in the pathogenesis of both NAFLD and HCC. NAFLD and NASH are associated with alterations in the gut microbial population through various routes, such as alterations in gut epithelial permeability, choline metabolism, endogenous alcohol production, release of inflammatory cytokines, regulation of hepatic TLR, and bile acid metabolism. In addition, altered bile acid metabolism, release of inflammatory cytokines, and TLR-4 expression may promote NAFLD/NASH-associated HCC. Potential targets for NAFLD treatment and for the prevention of HCC include composition of the microbiota, metabolites, and involved host pathways. However, better characterization of the intestinal microbiota and metabolome must be achieved through larger scale clinical research. Microbiota therapy could include the use of probiotic, prebiotic, and synbiotic supplements, or antibiotics. Conflict of interest The authors declare that they have no conflict of interest. Acknowledgments This study was supported by the USA National Institutes of Health (NIH) grant R01 AA020703, and by Award Number I01BX002213 from the Biomedical Laboratory Research & Development Service of the VA Office of Research and Development to B.Schnabl. References 1. Browning JD, Cohen JC, Hobbs HH. Patatin-like phospholipase domaincontaining 3 and the pathogenesis and progression of pediatric nonalcoholic fatty liver disease. Hepatology. 2010;52:1189e1192. 2. Chalasani N, Younossi Z, Lavine JE, et al. The diagnosis and management of non-alcoholic fatty fiver disease: practice guideline by the american gastroenterological association, american association for the study of liver diseases, and american college of gastroenterology. Gastroenterology. 2012;142: 1592e1609. 3. Kelly T, Yang W, Chen CS, Reynolds K, He J. Global burden of obesity in 2005 and projections to 2030. Int J Obes (Lond). 2008;32:1431e1437. 4. Caldwell S, Argo C. The natural history of non-alcoholic fatty liver disease. Dig Dis. 2010;28:162e168. 5. Michelotti GA, Machado MV, Diehl AM. NAFLD, NASH and liver cancer. Nat Rev Gastroenterol Hepatol. 2013;10:656e665. 6. Bhala N, Angulo P, van der Poorten D, et al. The natural history of nonalcoholic fatty liver disease with advanced fibrosis or cirrhosis: an international collaborative study. Hepatology. 2011;54:1208e1216.

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