Nonalcoholic Fatty Liver Disease: Basic Pathogenetic... : Transplantation (original) (raw)
Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease worldwide and includes a spectrum of histological features ranging from simple steatosis, characterized by fat accumulation in the liver, to nonalcoholic steatohepatitis (NASH), associated with ballooning of hepatocytes, inflammation, and/or fibrosis, finally driving to liver cirrhosis and hepatocellular carcinoma (HCC).1 Nonalcoholic fatty liver disease is estimated to affect about 25% of the general population, while NASH (ie, the evolutive form) has been calculated to be present in 2% to 5% of the general population.2 Cirrhosis and HCC in patients with NAFLD have been shown to represent the most growing indication for liver transplantation (OLTx).3
The first step in the development of NAFLD/NASH is represented by fat accumulation in the liver, a condition that is commonly associated with features of the metabolic syndrome (MetS), such as obesity, type 2 diabetes, dyslipidemia, and hypertension.2 From a pathological point of view, at least 3 mechanisms have been identified as source of excessive lipid accumulation into the liver, such as increased visceral adipose tissue (AT) lipolysis, hepatic de novo lipogenesis (DNL) activation and high content of calories and/or fat in the diet (Figure 1). Isotopes labeling has shown that the major mechanism is represented by excessive free fatty acids (FFAs) flux from the AT to the liver (59%), followed by DNL (26%%), and finally, by excess of calories and lipids in the diet (15%).4 Whereas simple steatosis is considered a more benign condition, NASH is associated with higher risk of cirrhosis and HCC development.5 Evolution from simple steatosis to NASH results from a complex interplay that involves either liver cell population (both parenchymal and nonparenchymal) and pathological signals coming from other organs, such as AT and the gut.6 Several pathological stimuli, including hepatocytes death, molecules secreted by AT, and intestinal pathogens can promote inflammation and fibrogenesis via the activation of resident macrophages (Kupffer cells [KCs]) that finally recruits monocytes and leukocytes from the circulation.7,8 This complex series of events finally culminate in the activation of hepatic stellate cells (HSCs), followed by the excessive synthesis and deposition of extracellular matrix.8
Pathogenesis of hepatic steatosis. Insulin resistance induces TG breakdown by the hydrolase activity of specific enzymes, such as ATGL, HSL, and MGL, which induces a flux of FFAs toward the liver. Furthermore, insulin resistance and the consequent hyperinsulinemia, can promote hepatic DNL via the transcriptional factor SREBP1c. Finally, dietary FAs, absorbed in the gut and incorporated as TGs into chylomicrons, can reach the liver to be accumulated. HSL, hormone-sensitive lipase; MGL, monoglyceride lipase.
Over time, several experimental models have been developed to mimic both metabolic as well as histologic features of NAFLD/NASH (Table 1). However, a model that could resemble all NAFLD features is still lacking. The challenge of researchers will be to establish the optimal animal model which could mirror human disease by providing the same pathological triggers, as well as reproducible mechanisms of progression toward NASH and its complications (ie, HCC development), to fully elucidate NAFLD pathogenesis and allow the development of effective therapies.
Animal models of NAFLD
So far, various pathological mechanisms that are associated with insulin-resistance and MetS participates in the pathogenesis and progression of NAFLD, including fat accumulation, lipotoxicity, oxidative stress, mitochondrial dysfunction, and alterations in the gut-liver axis signaling.6
MECHANISM OF FAT ACCUMULATION IN THE LIVER
Insulin resistance represents the key pathogenetic event associated with the development of hepatic steatosis. The status of insulin resistance makes AT resistant to the antilipolytic effect of insulin, leading to triglyceride (TG) breakdown and final formation of FFAs and glycerol.19 This enzymatic cleavage is allowed by the presence of specific enzymes with hydrolase activity, such as adipose TG lipase (ATGL), hormone-sensitive lipase, and monoglyceride lipase.20 Lack of AT lipolysis inhibition is thus associated with a massive release of FFAs that can be taken up by the liver where they accumulate as TG.21 In the presence of insulin resistance, higher insulin levels also modulate hepatic lipid metabolism by increasing TG synthesis.22 In the liver, DNL is a crucial pathway that contributes to lipid storage. Substrates coming from glycolysis (acetyl-CoA) initiate a multistep process that culminates in FFAs synthesis that are then converted into TG. De novo lipogenesis is under the control of 2 transcriptional factors, the sterol regulatory element binding protein 1c (SREBP1c) and the carbohydrate response element binding protein (ChREBP). SREBP1c activation occurs via 2 mechanisms that are, respectively, by insulin signaling.23 The first mechanism is mediated by the insulin receptor that signals the phosphoinositide-3 kinase/protein kinase B pathway.24 The second one is dependent by the nuclear receptor liver X receptor, in particular the hepatic isoform alpha, by phosphorylation of SREBP1c.25 Differently, ChREBP activation occurs after increased glucose concentration that enhances hepatic glycolysis. Intermediate and final products of glycolysis are thus able to activate ChREBP that regulates the expression of enzyme involved in lipogenesis, such as acetyl-CoA carboxylase, and fatty acid (FA) synthase.25
Dietary factors are obviously crucial for NAFLD development. High consumption of fat, typical of the so-called western diets, has been associated with insulin resistance, dyslipidemia, and metabolic/cardiovascular diseases.26 In addition, increased intake of sugar-sweetened food and beverages has been linked to NAFLD development. Different from glucose, fructose can modulate hepatic lipid metabolism either by the direct activation of SREBP1c and ChREBP, and by decreasing mitochondrial β-oxidation, finally favoring steatosis development.27,28 The key role of the western diet in the development of NAFLD has been demonstrated by the observation that lifestyle changes can strongly improve metabolic abnormalities, as well as hepatic steatosis and inflammation.29-31
LIPOTOXICITY, OXIDATIVE STRESS, AND MITOCHONDRIAL DYSFUNCTION
Accumulation of FFAs in the liver predisposes to lipotoxicity that promotes NASH development. Lipotoxicity is defined as dysregulation of the lipid environment and/or intracellular lipid composition, leading to accumulation of harmful lipids, which may be associated with organelle dysfunction, cell injury, and death. Lipotoxicity is intimately associated with chronic inflammation (metabolically triggered inflammation or metainflammation) associated with conditions predisposing to the progression from NAFLD to NASH, including obesity, diabetes, and MetS.6 Toxic lipids may cause cellular damage through 3 major mechanisms: (1) by modifying the biology and function of intracellular organelles, such as the endoplasmic reticulum (ER) and the mitochondria; (2) by directly modifying intracellular signaling pathways; (3) by interacting with specific proinflammatory cellular kinases, located at the cell surface or in the cytoplasm.
The ER is an intracellular component involved in protein assembly and its function is regulated by the unfolded protein response (UPR).32 Several stressors might alter ER homeostasis, associated with unfolded proteins production and accumulation that leads to UPR activation, as a rescue response. Unfolded protein response involves 3 intracellular pathways that can be activated by proteins localized in the ER membrane, such as RNA-dependent protein kinase-like ER eukaryotic initiation factor-2α kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring enzyme-1 (IRE1). Aim of UPR activation is the degradation of misfolded proteins, to maintain cell survival. However, when perpetuation of ER stress cannot be blocked/restored, Unfolded protein response leads to a series of pathological events.33 Concerning the specific role of ER stress in the progression from NAFLD to NASH, lipidomic analyses from human liver biopsies revealed increased levels of diglycerides and ceramides and of their transporter CD36 in steatotic livers.34 These lipids inhibit hepatic insulin signaling pathways, contributing to the onset of hepatic insulin resistance and the genesis of ER stress. The aberrant lipid changes in hepatocytes during hepatic steatosis can directly trigger chronic ER stress in the liver. Higher diacylglyceride, phospholipid, free cholesterol (FC), and FFA levels activate ER stress. Lipids can directly induce ER stress through IRE1 and PERK that sense the biophysical modifications of lipid membranes dependent on the ratio of unsaturated/saturated acyl chains. Saturated FFAs, such as palmitic acid (PA) or stearic acid, as opposed to unsaturated FFA, such as oleic acid, can become toxic for hepatocytes, activating through ER stress the mitochondrial pathway of apoptosis present in NASH pathogenesis by disrupting Ca2+ homeostasis.35-41
ER stress can induce hepatocyte apoptosis trough 3 ways: C/EBP homologous protein (CHOP) activation, c-Jun N-terminal kinase (JNK) signaling, and alteration of calcium homeostasis. Both PERK and ATF6 pathways induce CHOP activation, a transcription factor with well-known proapoptotic functions.42 The other branches of UPR are also strongly associated with cell death. Upon phosphorylation, IRE1 can bind the adaptor protein TNFα receptor-associated factor 2 (TRAF2) and promote apoptosis via JNK43 or by the direct activation of proapoptotic molecules, such as Bcl-2-like protein 4 and Bcl-2 homologous antagonist/killer.44 Finally, alteration of calcium homeostasis in ER lumen has been shown to drive ER stress and cell apoptosis.45
A cross-talk between extrinsic and intrinsic (endogenous) mitochondrial apoptosis pathways is a pathogenetic mechanism driving the progression from NAFLD to NASH.46 Tumor necrosis factor (TNF)-α–related apoptosis-inducing ligand (TRAIL) is a protein functioning as a ligand that induces the process of apoptosis.47 In the presence of nutrient excess, macrophage-associated hepatic and AT inflammation uses TRAIL receptor signaling to induce liver injury and fibrosis.48 On the other hand, incubation of primary hepatocytes or hepatocyte cell lines with PA or lysophosphatidylcholines increased the release of extracellular vesicles containing TRAIL that induced expression of IL1β in mouse bone marrow–derived macrophages.49 However, a clear role of TRAIL in mediating the progression from NAFLD to NASH is controversial. In the high-fat diet (HFD) model of NAFLD, TRAIL protects against insulin resistance, NAFLD development and vascular inflammation.50 In a nutritional model of NASH, again in Trail−/− mice, it was found that TRAIL deletion protected from liver injury, inflammation and fibrosis. In contrast to the liver, TRAIL deletion did not improve NASH-associated AT injury and inflammation, and aggravated insulin resistance, thus suggesting that liver-targeted inhibition of TRAIL signaling may be beneficial.51 To our knowledge, there are no clinical trials currently ongoing on TRAIL blockage.
Endoplasmic reticulum stress is also linked to chronic inflammation, through excessive production of reactive oxygen species (ROS) and the activation of nuclear factor kappa-light-chain-enhancer of activated B (NF-κB) and JNK pathways. There is a mutual, bidirectional interaction between ROS production and ER stress in driving the progression from NAFLD to NASH. Excessive FC in the liver of diabetic mice with NASH accumulates in mitochondria and ER, resulting in increased mitochondrial generation of ROS and apoptosis in a JNK1-dependent manner. These findings are dependent on the PA concentration to which hepatocytes were exposed.52 There is also a crosstalk between the ER and mitochondria. Exposure of primary hepatocytes to PA resulted in efflux of calcium from the ER leading to mitochondrial dysfunction and oxidative stress.53 One of the key mediator of mitochondrial dysfunction is generation of ROS during oxidative stress. CYP2E1 is implicated in hepatocyte injury and progression to NASH by promoting oxidative stress, inflammation, protein modification and insulin resistance.54 A significant amount of CYP2E1 is found in the mitochondria. This enzyme hydrolyzes various small molecules such as FFAs and ethanol into byproducts (toxic superoxide anion) that alter mitochondrial respiratory chain and damage mitochondrial constituents.55 Reactive oxygen species production can also promote hepatic inflammation by increasing the secretion of TNFα from hepatocytes and KCs, thus upregulating the synthesis of inflammatory cytokines.56 Activated IRE1 binds to TRAF2 that leads to the secretion of proinflammatory cytokines after NF-κB activation.43 Similarly, ER stress pathways mediated by PERK and ATF6 can promote inflammation45 (Figure 2).
ER stress in NAFLD/NASH pathogenesis. Three intracellular pathways that can be activated by membrane proteins localized in the ER membrane, as PERK, ATF6, and IRE1. In physiologic conditions, ER stress activation leads to misfolded proteins degradation to maintain its homeostasis. When the pathological stimuli is maintained over the time, such activation can trigger inflammation, oxidative stress and apoptosis. Concerning inflammation, both PERK and ATF6 activation can increase the secretion of proinflammatory cytokines via NF-κB. IRE1 pathway has also been demonstrated to mediate inflammation after TRAF2 binding. ROS synthesis induced by ER stress, can also promote hepatic inflammation by increasing the secretion of TNFα. ER stress can promote hepatocyte apoptosis. Although PERK and ATF6 pathways induce the activation of the proapoptotic transcription factor CHOP activation, IRE1 phosphorylation can promote apoptosis via JNK or Bax and Bak activation. Finally, alteration of calcium homeostasis in ER lumen has been shown to drive apoptosis.
Excess of FFAs can also directly trigger oxidative stress and ROS production independently from ER stress, and depending on catabolic processes, such as lipid oxidation. In normal conditions, FFAs are oxidized to carbon dioxide and water to produce energy and reduce lipid content in the liver. ROS concentration in such condition is usually minimal and this low level is strongly counteracted by the presence of efficient antioxidant systems.57 However, in the progression to NASH, 2 mechanisms induce oxidative stress. Free fatty acids overload upregulates mitochondrial and peroxisomal β-oxidation and increased ROS formation as a consequence, while, as a second mechanism, the endogenous antioxidant system is less efficient in the steatotic liver.58 Raised concentration of ROS can induce cell death in hepatocytes by activating specific pathways and inducing lipid peroxidation that results in the synthesis of reactive lipids, such as 4-hydroxy-2-nonenal and malondialdehyde. These reactive lipids derivatives can further amplify liver damage by promoting the release of ROS outside the hepatocytes contributing to HSCs activation and extracellular matrix deposition.59-63
A further pathogenetic event that is believed to be associated with NASH development is represented by mitochondrial dysfunction, although the mechanisms linking hepatic mitochondrial dysfunction with the progression from simple steatosis to NASH are largely unknown. The “remodeling” of mitochondrial energetics that includes several pathways, such as β-oxidation, hepatic tricarboxylic acid (TCA) cycle, and respiratory chain activity, is associated with NASH development64 (Figure 3). These pathways play a key role in the metabolic adaptation to FFAs overload, which occurs during NAFLD development, and are overexpressed in patients with NAFLD.65,66 Chronic production of acetyl-CoA through oxidative processes due to sustained FFAs flux can uncouple hepatic TCA cycle function from mitochondrial respiration, thus leading to impaired ATP synthesis and increased ROS generation.64 On this regard, it has been demonstrated that mitochondrial activity, which can be considered a protective mechanism to counteract lipid overload associated with oxidative processes, might be altered during NASH development.67 Furthermore, the incomplete β-oxidation, associated with excess of FFAs, can promote the synthesis and accumulation of toxic lipid intermediates, such as ceramides.68 Thus, manipulation of mitochondrial dysfunction seems to be a potential therapeutic approach to counteract NAFLD progression toward NASH. With this purpose, a mitochondrial uncoupler has been tested in an experimental model of NASH to improve both metabolic profile and the degree of liver injury.69 However, the use of these approaches in patients has not been studied.
Mitochondrial dysfunction. Several pathways, including β-oxidation, hepatic TCA cycle, and respiratory chain activity, seem to contribute to FFAs overload adaptation. However, in case of “chronic” lipid overload, these compensatory mechanisms can fail and become a source of toxic species, which can promote the progression of liver injury. Increased acetyl-CoA synthesis, as a product of FFAs β-oxidation, can uncouple hepatic TCA cycle function from mitochondrial respiration and promote ROS production. In addition to that, excess of substrate for β-oxidation (FFAs) might shift the normal metabolic process to the synthesis of toxic lipid intermediates, such as ceramides.
IDENTIFICATION OF TOXIC SPECIES
Hepatic TG accumulation can be considered the primum movens in the pathogenesis of NAFLD. However, TG accumulation is not associated with cell damage and represents a protective mechanism to counteract the progression of the disease associated with FFA-induced lipotoxicity.70–72 On this regard, ATGL inhibition, by reducing the release of FFAs from hepatic lipid droplets, has been found to protect against the progression of liver injury in mouse models of NASH.73
Free fatty acids can be classified according to number of carbon atoms, configuration of hydrogen atoms, and presence and position of bonds that confers different biological activities. Several in vitro experiments have highlighted a different role of saturated versus unsaturated FAs in inducing lipotoxicity.74-76 Many studies performed in hepatocytes cell lines have confirmed that exposure of saturated FAs, such as PA, leads to ER stress and cell death,38,77-79 and similar results have been shown in vivo.37,70,80 The toxic effect of saturated FFAs was also demonstrated by the inhibition of stearoyl-CoA desaturase-1, the enzyme responsible of fat desaturation, which was associated with increased cell death in a mouse model of NASH.70
Unsaturated FFAs are less toxic than PA, despite the fact that they contribute to steatosis, reinforcing the notion that fat accumulation and lipotoxicity are not synonymous.81 Moreover, these lipids may protect against cell death, reducing the levels of the proapoptotic proteins BIM (BCL2L11) and PUMA (BBC3), and favoring the sequestration of PA in TG.82 The specific mechanisms that explain such differences need to be further characterized but it is believed to be associated with the capacity of unsaturated FFAs to promote channeling of palmitate toward TG formation.74
Besides their role in lipotoxicity, saturated FFAs can promote hepatic inflammation by the induction of proinflammatory intracellular pathways mediated by toll-like receptor 4 (TLR4) activation that lead to increased TNFα production.33
Cholesterol has also been shown to contribute to NASH pathogenesis.70 The liver has specific mechanisms to counteract the accumulation of cholesterol, such as esterification of FC, control of its synthesis, and elimination regulated by the nuclear receptors farnesoid X receptor (FXR) and liver X receptor.83 However, in animal models of NASH, as well as in patients, increased amount of cholesterol, and in particular of FC, can be observed.83,84 In NASH, increased expression of SREBP-2 leads to upregulation of HMG-CoA reductase, the rate-limiting step of cholesterol synthesis, resulting in accumulation of FC, especially in the mitochondria.85 Mitochondrial FC deposition has been associated with hepatocytes death by JNK1 activation and TNFα production.86,87
In addition to its role in hepatocytes, FC can modify the behavior of nonparenchymal cells in the liver thus facilitating the progression from NAFLD to NASH. Free cholesterol can accumulate in KCs, in the form of cholesterol crystals, when they surround FC-enriched dead hepatocytes, leading to the synthesis of proinflammatory cytokines and chemokines.83 Hepatic stellate cells can also be affected by FC either as a consequence of hepatocytes and KCs activation or directly by signaling through a TLR4-dependent pathway.83
Ceramides can be also considered as toxic lipid products that drives both cell toxicity and inflammation. Ceramides may be generated de novo from serine and palmitoyl-CoA, via the enzyme serine palmitoyltransferase, or via the action of neutral sphingomyelinase, which catalyzes the release of ceramides from membrane sphingomyelins. Increased levels of ceramides have been reported in murine models and in patients with NASH.6 Harmful ceramides levels are associated with an increased amount of saturated fat in the diet in NAFLD patients.88 Generation of ceramides is increased by proinflammatory cytokines, including interleukin-1 and interleukin-6, and ceramides in turn contribute to inflammation via interaction with TNFα.89 Interference with ceramide synthesis was found to ameliorate steatosis, injury, and insulin sensitivity in rodent models of NAFLD, and the effects on steatosis may be linked to the induction of the FA transporter, CD3690 (Figure 4).
Toxic lipids. TGs are the main lipids stored in hepatocytes as droplets, and represent the hallmark of NAFLD. However, this form of accumulation is considered to protect against cell toxicity. Thus, monounsaturated FAs, which have been shown to be easily converted into TGs seems to protect the cells from lipotoxicity (A). On the other hand, saturated FAs, such as PA, are less prone to be esterified into neutral lipids (TGs) and are accumulated as FFAs. Therefore, this species can promote lipotoxicity (B). FC deposition in different liver cells (both parenchymal and nonparenchymal), has been also associated with lipotoxicity by promoting the release of proinflammatory cytokines and chemokines, thus facilitating the progression from NAFLD to NASH (B). Finally, ceramides, generated from serine and palmitoyl-CoA, via the enzyme serine palmitoyltransferase, or via the action of neutral sphingomyelinase, can trigger inflammation via interaction with TNFα (B).
DYSBIOSIS AND GUT-LIVER AXIS
The intestinal microbiota includes a large number of microorganisms, such as archeae, viruses, phages, yeast, and fungi. Included among them more than 1000 bacterial species, with a number of genes (the microbiome) that is hundreds of time higher than the human genome.91,92 The composition of gut microbiota differs along the different tracts of the intestine, and this is strictly associated with physiological conditions, such as gastrointestinal content flow rates, disposability of substrates for microorganisms, lumen pH, oxygen availability, and immunological features.93
In adult life, modifications of the gut microbiota can occur in relation to different diets, antibiotics use, and alcohol abuse.94 In healthy conditions, the gut flora maintains a symbiotic relationship with the host, and intestinal bacteria play a key role for immune system development and efficiency as well as for energy metabolism regulation.95 However, qualitative and quantity modifications of the gut microbiota, a condition called dysbiosis, can favor the development of chronic diseases, including NAFLD and its progression to NASH.96 Turnbaugh and his group97 have first discovered an important association between changes of gut microbiota composition and NAFLD, showing that the transfer of the gut microbiota from obese to lean rodents induced, in the recipient, the same metabolic alterations observed in the donors. Dysbiosis can induce NAFLD by several mechanisms (Figure 5). First, changes in gut microbiota have been shown to influence host metabolism by the synthesis of short-chain FAs (SCFAs). Short-chain FAs are metabolites of colonic bacterial fermentation of polysaccharides and includes acetate, propionate, and butyrate. In experimental models of NAFLD, as well as in obese patients, increased fecal SCFAs levels have been shown.97,98 Short-chain FAs can bind specific G-protein–coupled receptors, G-protein coupled receptor 41 (GPR41) and G-protein coupled receptors 43 (GPR43), that are expressed in all organs involved in the pathogenesis of NAFLD, such as AT, liver, and intestine. The activation of these receptors by the 3 different SCFAs can induce DNL, synthesis of cholesterol and alterations of glucose homeostasis.99 Furthermore, SCFAs can modulate food intake by regulating neuronal activity and thus appetite regulation.97 As a second major mechanism in inducing NAFLD, the gut microbiota can directly influence lipid metabolism. The most known effect is the capacity of intestinal bacteria to inhibit the synthesis of fasting-induced adipocyte factor (also known as angiopoietin-related protein 4), a specific inhibitor of the lipoprotein lipase (LPL). Lack of inhibition of LPL leads to a stronger release of FFAs from very low-density lipoprotein particles to the liver, favoring the development of steatosis.6
Role of dysbiosis and gut-liver axis in NAFLD. Diet-induced dysbiosis is associated with increased production of SCFAs. SCFAs can bind specific receptors, GPR41 and GPR43 and modulate DNL, cholesterol synthesis and glucose homeostasis, promoting hepatic and AT fat accumulation. In addition to that, dysbiosis, can reduce FIAF secretion from the intestine, an enzyme with inhibits LPS activity, thus leading to a higher release of FFAs from VLDL particles to the liver. Finally, modification of gut microbiota composition alters TJs expression, thus promoting the passage of bacteria/bacterial products into the portal circulation, facilitated by a less efficient AMPs secretion. Circulating bacteria or bacterial products can then lead to the development of liver inflammation and fibrosis. FIAF, fasting-induced adipocyte factor; VLDL, very low density lipoprotein.
A third key mechanism in the pathogenesis of NAFLD associated with dysbiosis is the alteration of the intestinal barrier, which favors the passage of bacteria or bacterial products such as lipopolysaccharide (LPS) into the portal circulation, thus leading to the progression from NAFLD to NASH as shown both in experimental and human studies.6,100,101 Translocation of bacteria or bacterial products is thus a key element in the progression from NAFLD to NASH and results from a failure in the defensive mechanisms present at the different levels in the intestinal wall. Regulation of intestinal barrier is the results of a complex interaction between microorganisms and mechanical and immunological mechanisms. The most important mechanical system is represented by the presence of tight junctions (TJ), multiprotein complexes that form a selectively permeable seal between adjacent epithelial cells.102 Diet-induced obesity alters the expression and distribution of TJ, and this is directly associated with increased intestinal permeability.100,103-105 A direct role of dysbiosis was also confirmed by the fact that gut decontamination restored the efficacy of the intestinal barrier. A different defensive mechanism include the secretion of several molecules involved in bacterial killing, the antimicrobial peptides (AMPs), and the secretion of cytokines by specific cells such as lymphoid cells, macrophages and neutrophils. Dysbiosis driven by HFD reduces the expression of AMPs, and this is associated with NASH development.105,106
The presence of a complex immune system in the intestine is also crucial for the maintenance of a proper tolerance to gut microbiota. The innate immune response is regulated by pattern recognition receptors, such as transmembrane receptors and cytosolic nucleotide-binding oligomerization domain (NOD)-like receptors (NLRPs) that are sensed by intestinal microorganisms. Among the adaptive immune system, secretion of IgA by B cells represents an important mechanism in the control of gut microbiota.107 Whereas in normal conditions, the immunological response participates in the control of bacterial growth and pathogenicity, the presence of dysbiosis can lead to alterations in this cross-talk between the immune system and the gut.107 When the mechanisms involved in the regulation of gut barrier are altered, bacterial translocation occurs and provides pathogenetic signals to several organs, including the liver. Specifically bacterial components can increase the expression of specific receptor such as TLRs. Toll-like receptors are pattern recognition receptors of the innate immune response that can sense bacterial products. TLRs activation is associated with a proinflammatory response mediated by tumor necrosis factor (TNF)-α, interleukin-1β (IL-1β), and interferons. Among the 13 TLRs identified in mammals, the pathogenesis of NAFLD/NASH is associated with TLR2, TLR4, and TLR9, which recognize LPS, peptidoglycans, flagellins, and bacterial DNA, respectively.108 In addition to bacterial products, FFAs also can directly activate TLR4.33 The association between TLRs and NAFLD has been extensively demonstrated in both animal models, as well as in human.109-113 Increased levels of LPS, a Gram-negative bacterial wall component that activates TLR4, has been observed in the serum of NASH patients and in experimental murine models of NAFLD, such as the HFD, and NASH, such as fructose-rich diet, methionine/choline-deficient diet, and choline-deficient amino acid-defined diet and lack of TLR4 has been shown to reduce liver damage, in terms of both steatosis and steatohepatitis.110,114-116
Dysbiosis has been also associated with the development of HCC,117 as shown by the observation that microbiota modulation, either via gut sterilization or probiotic treatment, reduced HCC formation and growth in murine experimental models.117-119 However, these articles provided conflicting results indicating either the role of Gram-negative117 or Gram-positive bacteria119 as main players of the oncogenetic process. These differences are presumably related to the different experimental models used and to the different breeding conditions. On the other hand, these studies indicate a key role of gut-derived signaling in activating nonparenchymal liver cells, such as KCs and HSCs, in promoting proinflammatory and survival signals. In a series of NAFLD cirrhotic patients with HCC, enhanced intestinal inflammatory status has been demonstrated compared to those without HCC and healthy subjects. Furthermore, the degree of intestinal inflammation was correlated with humoral and cellular parameters of systemic inflammation, and this inflammatory landscape was influenced by the particular composition of the gut microbiota.120
On the basis of its important contribution to liver injury progression and hepatocarcinogenesis, the gut-microbiota-liver axis represents a promising target to treat NAFLD and its progression to NASH and HCC. Preclinical studies show a drastic (~80%) reduction of HCC development in mouse models.117-119 In addition to potentially reducing the risk of HCC development, targeting the gut-microbiota-liver axis has been shown to reduce liver fibrosis103,105 and portal hypertension121 in rodents, and spontaneous bacterial peritonitis122 and hepatic encephalopathy123 in patients. With the increased understanding of the underlying pathophysiology of NAFLD/NASH, the number of clinically feasible approaches to target the gut-microbiota-liver axis is continuously growing and includes the use of antibiotics, probiotics, therapies that target the gut barrier, the transplantation of microbiota, and the use of TLRs antagonists.6
However, although based on strong experimental data, the clinical translation of these data is reduced by the potential side effects of these therapies. One example is represented by increased production of TNFα by either monocytes in mesenteric lymph nodes, representing one of the main factors increasing TJ permeability, or KCs in the liver amplifying the degree of liver injury.6 However, the lesson we learned from the treatment of alcoholic hepatitis indicates that translating these findings to patients might be difficult because of strong immunosuppressive effects of TNF inhibitors and increased rates of severe infection.124,125 Owing to these adverse effects, long-term anti-TNFα therapy in patients with chronic liver disease might confer more harm than benefit.
On the other hand, the FXR agonist obeticholic acid (OCA) attenuates mucosal injury, ileal barrier permeability, bacterial overgrowth, and bacterial translocation in experimental models of liver injury126,127 whereas OCA has shown a high safety profile in patients with NASH as demonstrated in the FLINT trial, with the major adverse effects being pruritus and alterations of serum lipid profiles.128 A phase 3 trial is now ongoing.
Many of the hepatic effects of FXR activation are mediated by intestinal FXR receptors, resulting in the release of FGF19 that acts on targets in the liver.129 The use of FGF19 as a therapy for NASH, however, is hampered by its oncogenic properties.130,131 NGM282 is an FGF19 analog that, unlike FGF19, does not activate signal transducer and activator of transcription 3, a signaling pathway essential for FGF19-mediated hepatocarcinogenesis. In animal models of nonalcoholic steatohepatitis, treatment with NGM282 resulted in a rapid and robust reduction in concentrations of alanine aminotransferase and aspartate aminotransferase, as well as a clear improvement in all histological features associated with nonalcoholic steatohepatitis, including hepatic steatosis, inflammation, ballooning degeneration, and fibrosis.132 In a randomized, double-blind, placebo-controlled, phase 2 study, NGM282 produced rapid and significant reductions in liver fat content with an acceptable safety profile in patients with nonalcoholic steatohepatitis.133
Although several clinical trial are ongoing concerning microbiota manipulation, with pre and/or probiotics, no significant clinical data have been published so far. Microbiota manipulation can be also obtained by fecal microbiota transplantation (FMT). Fecal microbiota transplantation is now part of the therapeutic options for Clostridium difficile infection. A randomized controlled trial demonstrated amelioration of hepatic and peripheral insulin resistance in patients with MetS who had received fecal microbiota from lean donors.134 However, differently from patients with C. difficile infection, there are concerns regarding the use of FMT in patients with chronic liver diseases due to the number of treatments, the modifications of microbiota during treatment, and the transmission of pathogens during FMT therapy. Ideally, feces might be substituted in favor of defined mixtures of cultured bacteria that resemble the human microbiota transplanted via FMT and confer the same beneficial effects, being FMT more acceptable to patients and physicians. Once this goal has been achieved, patients with chronic liver disease should be considered as potential candidates to study the effect of FMT on disease progression.
INFLAMMASOME AND NAFLD
Toll-like receptor signaling can also act via the activation of inflammasomes. Inflammasomes are multiprotein complexes localized in the cytoplasm that can be activated by either exogenous pathogen-associated molecular patterns, such as LPS, or internal host damage-associated molecular patterns.135 The ligand membrane receptors, activate a complex intracellular cascade that results in the secretion of IL-1β and IL-18, which have proinflammatory and profibrotic effects. Recently, the expression of inflammasome components was demonstrated to be increased in animal models and in patients with NASH, indicating its potential role in the progression of liver injury, although the deletion of specific inflammasome components has shown no univocal effects on the development and progression of liver injury.105,136-140 Overall inflammasome activation in the liver is, however, a key element in the gut-liver axis cross-talk because, by receiving pathogenetic signaling from the intestine, it drives the progression from NAFLD to NASH and HCC.16 The mechanisms behind the effects of the inflammasome in NASH development are complexes and might involve alterations in metabolic, inflammatory, and profibrogenic pathways in the liver (parenchymal and nonparenchymal cells) and AT, immune system. The inflammasome components in the gut can also strongly affect the progression from NAFLD to NASH, because the deletion of inflammasome components was associated with dysbiosis and pathobionts selection that lead to intestinal mucus degradation, loss of epithelial integrity, gut inflammation, bacteremia, and a more severe degree of liver injury as also demonstrated by cohousing experiments.137 To confirm this, a western lifestyle diet induced NASH only if associated with lack of NLRP3-inflammasome. These effects were associated with the expansion of profibrogenetic Gram-negative proteobacteria and verrucomicrobia,105 which, together with verrucomicrobia, are mucus-degrading bacteria which can favor bacterial translocation (Figure 6).
Inflammasome and NAFLD. The association between a defective innate immune response and a Western-lifestyle diet leads to the expansion of a pathogenetic microbiota, with abundance of Gram-negative species and gut barrier alterations (as reduced AMPs activity). These conditions promote BT that drives a worsened NASH phenotype via 2 mechanisms. Pathogens can trigger AT inflammation which impairs insulin signaling and promotes the release of FFAs that can be taken up by the liver. In addition, Gram-negative bacteria can activate proinflammatory pathways and oxidative stress in the liver, following TLRs binding. BT, bacterial translocation.
IMMUNE DYSREGULATION AND CHRONIC INFLAMMATION IN NASH
The current view indicates that innate immune response, starting from gut signals and translated to the liver by specific receptors, such as TLRs, represents the main factor in the onset of hepatic inflammation in NASH, leading to the activation of KCs that release a variety of proinflammatory cytokines and chemokines.6 However, several data on the specificity of chronic inflammation can also have a role in the progression of NASH to cirrhosis. One of the histological features of NASH is a diffuse lobular infiltration by lymphocytes and macrophages141 that are also the most frequent inflammatory cells in periportal infiltrates of NASH.142 In parallel with the worsening of parenchymal injury and lobular inflammation, CD4+ and CD8+ T-, B lymphocytes and natural killer T cells (NKT) are recruited within the liver.143-145 These T lymphocytes express the activation markers CD44 and CD69, along with an enhanced production of IFN-γ and TNF superfamily member 14 (TNFSF14), indicating that liver infiltrating lymphocytes are functionally activated.143,145 Circulating vascular adhesion protein-1, an amino-oxidase constitutively expressed on human hepatic endothelium, is increased in patients with NASH as compared to those with NAFLD or healthy controls and it associates with an enhanced severity of hepatic inflammation and fibrosis.144 Liver recruitment of CD4+ T- and NKT cells in NASH specifically involves an increase in the hepatic production of vascular adhesion protein-1.144 Furthermore, the choline-deficient model of NASH can progress to HCC and this is characterized by increased inflammasome activation.16 However, it has also been shown that in this model steatosis, parenchymal injury, and lobular inflammation are greatly lowered without tumor formation in Rag1−/− mice lacking mature B, T, and NKT cells and unable to mount adaptive immune responses. The prevention of hepatic steatosis in Rag1−/− mice appears unrelated to metabolic abnormalities, insulin resistance or changes in gut microbiota, but depends on the direct interaction between hepatocytes and NKT cells.145 The clinical relevance of these findings is supported by observations showing that both pediatric and adult NASH are characterized by an increase in circulating IFN-γ-producing CD4+ and CD8+ T lymphocytes, whereas also upregulation of Th-17 related cytokines, such as IL-17, IL-21, and IL-23, was observed in liver biopsies of NASH patients.146-148 Although these findings have several analogies with those observed in obesity where CD4+/CD8+ T lymphocytes recruited to the AT support macrophage production of proinflammatory mediators,149 they also underline the role of immune reactions and chronic inflammation in playing a role in NASH progression to the most advanced stage of liver disease.
Because our knowledge on the immune mechanisms supporting steatohepatitis in NASH are still quite preliminary, only few attempts have been made to exploits the therapeutic possibility of interfering with the immune system. In addition to several experimental approaches,144,150-152 oral OKT3 antibodies to block lymphocytes T-cell receptor function have been reported to ameliorate liver enzyme release and blood glucose and insulin levels when tested in patients with NASH and altered glucose metabolism or type-2 diabetes using a single-blind randomized placebo-controlled phase-2 study.153
In NASH, and differently from NAFLD, the inflammatory infiltrate and KCs activation are associated with overexpression of proinflammatory chemokines, such as CCL2 (MCP1) and CCL5 (RANTES), that contributes to activation and migration of inflammatory cells into the liver, as well as to the progression of fibrosis.6 Cenicriviroc is an oral antagonist of CCR2/CCR5, the chemokine receptors for MCP1 and RANTES, respectively, in both activated KCs and inflammatory monocytes. Cenicriviroc has been tested in a randomized, double-blind, multinational phase 2b study enrolling subjects with NASH. The primary endpoint of histological improvement (>2-point in the NAS score) in the intent-to-treat population and resolution of NASH was achieved in a similar proportion of treated subjects compared with placebo, but the fibrosis endpoint (improvement in fibrosis stage by >1 stage and no worsening of NASH) was met in significantly more subjects on Cenicriviroc than placebo.154 A phase-3 trial is now ongoing.
NAFLD AND NASH RECURRENCE POST-OLTX
Nonalcoholic steatohepatitis is becoming a major indication for OLTx.155 It has been shown that patient survival after liver transplantation in patients who underwent transplantation for cryptogenic cirrhosis or NASH is similar compared with those patients who underwent transplantation for all other indications.156,157 About 50% of patients who underwent transplantation for NASH have NAFLD recurrence post-OLTx, and 75% of these patients have NASH, although advanced fibrosis occurs in less than 10% of patients.156,158 Theoretically, there are donor-, recipient-, and transplant-specific factors that could contribute to de novo or recurrent NAFLD and NASH after OLTx; however, little is known concerning the pathogenesis of these conditions.
Concerning the donor-specific factors, there is no known association between donor clinical risk factors for NAFLD or NASH and the risk for de novo or recurrent hepatic steatosis in the transplant recipients. The limited use of donor allografts known to have hepatic steatosis and the lack of surveillance or protocol post-OLTx liver biopsies did not allow to have data useful in defining the prevalence, incidence, and/or natural history of donor hepatic steatosis on de novo or recurrent NAFLD and the associated transplant outcomes.
Concerning the recipient-specific factors, it is not known whether sex and age play a role in the risk prevalence of de novo or recurrent NAFLD after OLTx. The ethnic differences in the prevalence of NAFLD and NASH pretransplant (white persons > Latinos > African Americans) may account for the reported variance in incidence across ethnicities of de novo and recurrent NAFLD post-OLTx.159 Post-OLTx rapid weight gain and MetS components (hypertension, diabetes, and hyperlipidemia) occur in a large proportion of patients but their specific role on de novo or recurrent NAFLD and NASH is not known.160
Aside from the multitude of metabolic risk factors that place patients at risk for the development of de novo or recurrent NAFLD, immunosuppression can lead to or exacerbate metabolic risk factors for NAFLD. In addition to affecting fat metabolism and storage, glucocorticoids also contribute to hypertension, dyslipidemia, and insulin resistance.161,162 Specific data on the post-OLTx setting are lacking, and corticosteroids are a necessary component of the post-OLTx immunosuppression strategy; however, they are associated with a pharmacological profile that should be recognized as a risk factor for developing de novo NAFLD. Although highly effective in minimizing risk for cellular rejection, CNIs are also associated with hypertension, hyperlipidemia, new-onset diabetes, and chronic renal disease after liver transplantation. CNI withdrawal and replacement with mycophenolate mofetil has been shown to improve post-OLTx dyslipidemia.163 Also sirolimus and everolimus, as part of the mammalian target of rapamycin inhibitors family, increases TG production, and alters insulin signaling pathways, thus leading to metabolic alterations in liver, muscle, and AT, which may contribute to the development of dyslipidemia and insulin resistance and therefore confer increased risk for de novo or recurrent NAFLD.164,165
Thus, de novo or recurrent post-OLTx steatosis lacks of most of the pathogenetic knowledge that have been firmly established in “naive” NAFLD/NASH and only association data are known.
CONCLUSIONS
Despite the advances in understanding the mechanisms responsible for NAFLD development and its progression to NASH, many aspects still need to be clarified. The lack of knowledge regarding such mechanisms can also explain the lack of effective therapies availability so far.
In this review, we addressed the most important pathogenetic processes involved in the development of NAFLD and NASH. The role of insulin resistance is strongly correlated with fat accumulation because it can contribute to the release of FFAs from AT and directly influence hepatic lipid metabolism. Lipid metabolism is also modulated by intestinal products that derive from bacteria fermentation of polysaccharides in the course of dysbiosis. Moreover, dysbiosis and translocation of bacterial product drive the progression from NAFLD to NASH by activating proinflammatory and profibrogenetic intracellular pathways mediated by TLRs and inflammasome activation.
REFERENCES
1. Sayiner M, Koenig A, Henry L, et al. Epidemiology of nonalcoholic fatty liver disease and nonalcoholic steatohepatitis in the United States and the rest of the world. Clin Liver Dis. 2016;20:205–214.
2. Younossi ZM, Koenig AB, Abdelatif D, et al. Global epidemiology of nonalcoholic fatty liver disease-meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology. 2016;64:73–84.
3. Pais R, Barritt AS 4th, Calmus Y, et al. NAFLD and liver transplantation: current burden and expected challenges. J Hepatol. 2016;65:1245–1257.
4. Donnelly KL, Smith CI, Schwarzenberg SJ, et al. Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty liver disease. J Clin Invest. 2005;115:1343–1351.
5. Dulai PS, Singh S, Patel J, et al. Increased risk of mortality by fibrosis stage in nonalcoholic fatty liver disease: systematic review and meta-analysis. Hepatology. 2017;65:1557–1565.
6. Marra F, Svegliati-Baroni G. Lipotoxicity and the gut-liver axis in NASH pathogenesis. J Hepatol. 2018;68:280–295.
7. Wolfs MG, Gruben N, Rensen SS, et al. Determining the association between adipokine expression in multiple tissues and phenotypic features of non-alcoholic fatty liver disease in obesity. Nutr Diabetes. 2015;5:e146.
8. Koyama Y, Brenner DA. Liver inflammation and fibrosis. J Clin Invest. 2017;127:55–64.
9. Lau JK, Zhang X, Yu J. Animal models of non-alcoholic fatty liver disease: current perspectives and recent advances. J Pathol. 2017;241:36–44.
10. Hebbard L, George J. Animal models of nonalcoholic fatty liver disease. Nat Rev Gastroenterol Hepatol. 2011;8:35–44.
11. Takahashi Y, Soejima Y, Fukusato T. Animal models of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. World J Gastroenterol. 2012;18:2300–2308.
12. Matsuzawa N, Takamura T, Kurita S, et al. Lipid-induced oxidative stress causes steatohepatitis in mice fed an atherogenic diet. Hepatology. 2007;46:1392–1403.
13. Lebeaupin C, Vallee D, Hazari Y, et al. Endoplasmic reticulum stress signalling and the pathogenesis of non-alcoholic fatty liver disease. J Hepatol. 2018;69:927–947.
14. Corbin KD, Zeisel SH. Choline metabolism provides novel insights into nonalcoholic fatty liver disease and its progression. Curr Opin Gastroenterol. 2012;28:159–165.
15. Rinella ME, Green RM. The methionine-choline deficient dietary model of steatohepatitis does not exhibit insulin resistance. J Hepatol. 2004;40:47–51.
16. De Minicis S, Agostinelli L, Rychlicki C, et al. HCC development is associated to peripheral insulin resistance in a mouse model of NASH. PloS One. 2014;9:e97136.
17. Matsumoto M, Hada N, Sakamaki Y, et al. An improved mouse model that rapidly develops fibrosis in non-alcoholic steatohepatitis. Int J Exp Pathol. 2013;94:93–103.
18. Castro RE, Diehl AM. Towards a definite mouse model of NAFLD. J Hepatol. 2018;69:272–274.
19. Schweiger M, Romauch M, Schreiber R, et al. Pharmacological inhibition of adipose triglyceride lipase corrects high-fat diet-induced insulin resistance and hepatosteatosis in mice. Nat Commun. 2017;8:14859.
20. Schweiger M, Schreiber R, Haemmerle G, et al. Adipose triglyceride lipase and hormone-sensitive lipase are the major enzymes in adipose tissue triacylglycerol catabolism. J Biol Chem. 2006;281:40236–40241.
21. Samuel VT, Shulman GI. Mechanisms for insulin resistance: common threads and missing links. Cell. 2012;148:852–871.
22. Vatner DF, Majumdar SK, Kumashiro N, et al. Insulin-independent regulation of hepatic triglyceride synthesis by fatty acids. Proc Natl Acad Sci U S A. 2015;112:1143–1148.
23. Kawano Y, Cohen DE. Mechanisms of hepatic triglyceride accumulation in non-alcoholic fatty liver disease. J Gastroenterol. 2013;48:434–441.
24. Sanders FW, Griffin JL. De novo lipogenesis in the liver in health and disease: more than just a shunting yard for glucose. Biol Rev Camb Philos Soc. 2016;91:452–468.
25. Beaven SW, Matveyenko A, Wroblewski K, et al. Reciprocal regulation of hepatic and adipose lipogenesis by liver X receptors in obesity and insulin resistance. Cell Metab. 2013;18:106–117.
26. Fan JG, Cao HX. Role of diet and nutritional management in non-alcoholic fatty liver disease. J Gastroenterol Hepatol. 2013;28(Suppl 4):81–87.
27. Kennedy AR, Pissios P, Otu H, et al. A high-fat, ketogenic diet induces a unique metabolic state in mice. Am J Physiol Endocrinol Metab. 2007;292:E1724–E1739.
28. Caligiuri A, Gentilini A, Marra F. Molecular pathogenesis of NASH. Int J Mol Sci. 2016;17.
29. Wong VW, Chan RS, Wong GL, et al. Community-based lifestyle modification programme for non-alcoholic fatty liver disease: a randomized controlled trial. J Hepatol. 2013;59:536–542.
30. Rodríguez-Piñeiro AM, Johansson ME. The colonic mucus protection depends on the microbiota. Gut Microbes. 2015;6:326–330.
31. Romero-Gomez M, Zelber-Sagi S, Trenell M. Treatment of NAFLD with diet, physical activity and exercise. J Hepatol. 2017;67:829–846.
32. Kaufman RJ. Orchestrating the unfolded protein response in health and disease. J Clin Invest. 2002;110:1389–1398.
33. Fuchs M, Sanyal AJ. Lipotoxicity in NASH. J Hepatol. 2012;56:291–293.
34. Gorden DL, Myers DS, Ivanova PT, et al. Biomarkers of NAFLD progression: a lipidomics approach to an epidemic. J Lipid Res. 2015;56:722–736.
35. Fu S, Yang L, Li P, et al. Aberrant lipid metabolism disrupts calcium homeostasis causing liver endoplasmic reticulum stress in obesity. Nature. 2011;473:528–531.
36. Hirsova P, Ibrahim SH, Gores GJ, et al. Lipotoxic lethal and sublethal stress signaling in hepatocytes: relevance to NASH pathogenesis. J Lipid Res. 2016;57:1758–1770.
37. Wang D, Wei Y, Pagliassotti MJ. Saturated fatty acids promote endoplasmic reticulum stress and liver injury in rats with hepatic steatosis. Endocrinology. 2006;147:943–951.
38. Pfaffenbach KT, Gentile CL, Nivala AM, et al. Linking endoplasmic reticulum stress to cell death in hepatocytes: roles of C/EBP homologous protein and chemical chaperones in palmitate-mediated cell death. Am J Physiol Endocrinol Metab. 2010;298:E1027–E1035.
39. Cazanave SC, Mott JL, Bronk SF, et al. Death receptor 5 signaling promotes hepatocyte lipoapoptosis. J Biol Chem. 2011;286:39336–39348.
40. Volmer R, van der Ploeg K, Ron D. Membrane lipid saturation activates endoplasmic reticulum unfolded protein response transducers through their transmembrane domains. Proc Natl Acad Sci U S A. 2013;110:4628–4633.
41. Deniaud A, Sharaf el dein O, Maillier E, et al. Endoplasmic reticulum stress induces calcium-dependent permeability transition, mitochondrial outer membrane permeabilization and apoptosis. Oncogene. 2008;27:285–299.
42. Tabas I, Ron D. Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress. Nat Cell Biol. 2011;13:184–190.
43. Urano F, Wang X, Bertolotti A, et al. Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1. Science. 2000;287:664–666.
44. Hetz C, Bernasconi P, Fisher J, et al. Proapoptotic BAX and BAK modulate the unfolded protein response by a direct interaction with IRE1alpha. Science. 2006;312:572–576.
45. Zhang XQ, Xu CF, Yu CH, et al. Role of endoplasmic reticulum stress in the pathogenesis of nonalcoholic fatty liver disease. World J Gastroenterol. 2014;20:1768–1776.
46. Farrell GC, Larter CZ, Hou JY, et al. Apoptosis in experimental NASH is associated with p53 activation and TRAIL receptor expression. J Gastroenterol Hepatol. 2009;24:443–452.
47. Wiley SR, Schooley K, Smolak PJ, et al. Identification and characterization of a new member of the TNF family that induces apoptosis. Immunity. 1995;3:673–682.
48. Idrissova L, Malhi H, Werneburg NW, et al. TRAIL receptor deletion in mice suppresses the inflammation of nutrient excess. J Hepatol. 2015;62:1156–1163.
49. Hirsova P, Ibrahim SH, Krishnan A, et al. Lipid-induced signaling causes release of inflammatory extracellular vesicles from Hepatocytes. Gastroenterology. 2016;150:956–967.
50. Cartland SP, Harith HH, Genner SW, et al. Non-alcoholic fatty liver disease, vascular inflammation and insulin resistance are exacerbated by TRAIL deletion in mice. Sci Rep. 2017;7:1898.
51. Hirsova P, Weng P, Salim W, et al. TRAIL deletion prevents liver, but not adipose tissue, inflammation during murine diet-induced obesity. Hepatol Commun. 2017;1:648–662.
52. Win S, Than TA, Le BH, et al. Sab (Sh3bp5) dependence of JNK mediated inhibition of mitochondrial respiration in palmitic acid induced hepatocyte lipotoxicity. J Hepatol. 2015;62:1367–1374.
53. Egnatchik RA, Leamy AK, Noguchi Y, et al. Palmitate-induced activation of mitochondrial metabolism promotes oxidative stress and apoptosis in H4IIEC3 rat hepatocytes. Metabolism. 2014;63:283–295.
54. Abdelmegeed MA, Banerjee A, Yoo SH, et al. Critical role of cytochrome P450 2E1 (CYP2E1) in the development of high fat-induced non-alcoholic steatohepatitis. J Hepatol. 2012;57:860–866.
55. Aubert J, Begriche K, Knockaert L, et al. Increased expression of cytochrome P450 2E1 in nonalcoholic fatty liver disease: mechanisms and pathophysiological role. Clin Res Hepatol Gastroenterol. 2011;35:630–637.
56. Kern PA, Saghizadeh M, Ong JM, et al. The expression of tumor necrosis factor in human adipose tissue. Regulation by obesity, weight loss, and relationship to lipoprotein lipase. J Clin Invest. 1995;95:2111–2119.
57. Neuschwander-Tetri BA. Hepatic lipotoxicity and the pathogenesis of nonalcoholic steatohepatitis: the central role of nontriglyceride fatty acid metabolites. Hepatology. 2010;52:774–788.
58. Yesilova Z, Yaman H, Oktenli C, et al. Systemic markers of lipid peroxidation and antioxidants in patients with nonalcoholic fatty liver disease. Am J Gastroenterol. 2005;100:850–855.
59. Yu J, Marsh S, Hu J, et al. The pathogenesis of nonalcoholic fatty liver disease: interplay between diet, gut microbiota, and genetic background. Gastroenterol Res Pract. 2016;2016:2862173.
60. Galli A, Svegliati-Baroni G, Ceni E, et al. Oxidative stress stimulates proliferation and invasiveness of hepatic stellate cells via a MMP2-mediated mechanism. Hepatology. 2005;41:1074–1084.
61. Svegliati-Baroni G, Candelaresi C, Saccomanno S, et al. A model of insulin resistance and nonalcoholic steatohepatitis in rats: role of peroxisome proliferator-activated receptor-alpha and n-3 polyunsaturated fatty acid treatment on liver injury. Am J Pathol. 2006;169:846–860.
62. De Minicis S, Candelaresi C, Agostinelli L, et al. Endoplasmic reticulum stress induces hepatic stellate cell apoptosis and contributes to fibrosis resolution. Liver Int. 2012;32:1574–1584.
63. Svegliati-Baroni G, Saccomanno S, van Goor H, et al. Involvement of reactive oxygen species and nitric oxide radicals in activation and proliferation of rat hepatic stellate cells. Liver. 2001;21:1–12.
64. Sunny NE, Bril F, Cusi K. Mitochondrial adaptation in nonalcoholic fatty liver disease: novel mechanisms and treatment strategies. Trends Endocrinol Metab. 2017;28:250–260.
65. Sunny NE, Parks EJ, Browning JD, et al. Excessive hepatic mitochondrial TCA cycle and gluconeogenesis in humans with nonalcoholic fatty liver disease. Cell Metab. 2011;14:804–810.
66. Iozzo P, Bucci M, Roivainen A, et al. Fatty acid metabolism in the liver, measured by positron emission tomography, is increased in obese individuals. Gastroenterology. 2010;139:846–856, 856.e841–e846.
67. Koliaki C, Szendroedi J, Kaul K, et al. Adaptation of hepatic mitochondrial function in humans with non-alcoholic fatty liver is lost in steatohepatitis. Cell Metab. 2015;21:739–746.
68. Patterson RE, Kalavalapalli S, Williams CM, et al. Lipotoxicity in steatohepatitis occurs despite an increase in tricarboxylic acid cycle activity. Am J Physiol Endocrinol Metab. 2016;310:E484–E494.
69. Perry RJ, Zhang D, Zhang XM, et al. Controlled-release mitochondrial protonophore reverses diabetes and steatohepatitis in rats. Science. 2015;347:1253–1256.
70. Alkhouri N, Dixon LJ, Feldstein AE. Lipotoxicity in nonalcoholic fatty liver disease: not all lipids are created equal. Expert Rev Gastroenterol Hepatol. 2009;3:445–451.
71. Yamaguchi K, Yang L, McCall S, et al. Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis. Hepatology. 2007;45:1366–1374.
72. Papazyan R, Sun Z, Kim YH, et al. Physiological suppression of lipotoxic liver damage by complementary actions of HDAC3 and SCAP/SREBP. Cell Metab. 2016;24:863–874.
73. Fuchs CD, Claudel T, Kumari P, et al. Absence of adipose triglyceride lipase protects from hepatic endoplasmic reticulum stress in mice. Hepatology. 2012;56:270–280.
74. Listenberger LL, Ory DS, Schaffer JE. Palmitate-induced apoptosis can occur through a ceramide-independent pathway. J Biol Chem. 2001;276:14890–14895.
75. Maedler K, Spinas GA, Dyntar D, et al. Distinct effects of saturated and monounsaturated fatty acids on beta-cell turnover and function. Diabetes. 2001;50:69–76.
76. de Vries JE, Vork MM, Roemen TH, et al. Saturated but not mono-unsaturated fatty acids induce apoptotic cell death in neonatal rat ventricular myocytes. J Lipid Res. 1997;38:1384–1394.
77. Cao J, Dai DL, Yao L, et al. Saturated fatty acid induction of endoplasmic reticulum stress and apoptosis in human liver cells via the PERK/ATF4/CHOP signaling pathway. Mol Cell Biochem. 2012;364:115–129.
78. Leamy AK, Egnatchik RA, Shiota M, et al. Enhanced synthesis of saturated phospholipids is associated with ER stress and lipotoxicity in palmitate treated hepatic cells. J Lipid Res. 2014;55:1478–1488.
79. Egnatchik RA, Leamy AK, Jacobson DA, et al. ER calcium release promotes mitochondrial dysfunction and hepatic cell lipotoxicity in response to palmitate overload. Mol Metab. 2014;3:544–553.
80. Li J, Huang J, Li JS, et al. Accumulation of endoplasmic reticulum stress and lipogenesis in the liver through generational effects of high fat diets. J Hepatol. 2012;56:900–907.
81. Li ZZ, Berk M, McIntyre TM, et al. Hepatic lipid partitioning and liver damage in nonalcoholic fatty liver disease: role of stearoyl-CoA desaturase. J Biol Chem. 2009;284:5637–5644.
82. Akazawa Y, Cazanave S, Mott JL, et al. Palmitoleate attenuates palmitate-induced Bim and PUMA up-regulation and hepatocyte lipoapoptosis. J Hepatol. 2010;52:586–593.
83. Ioannou GN. The role of cholesterol in the pathogenesis of NASH. Trends Endocrinol Metab. 2016;27:84–95.
84. Wouters K, van Bilsen M, van Gorp PJ, et al. Intrahepatic cholesterol influences progression, inhibition and reversal of non-alcoholic steatohepatitis in hyperlipidemic mice. FEBS Lett. 2010;584:1001–1005.
85. Caballero F, Fernandez A, De Lacy AM, et al. Enhanced free cholesterol, SREBP-2 and StAR expression in human NASH. J Hepatol. 2009;50:789–796.
86. Gan LT, Van Rooyen DM, Koina ME, et al. Hepatocyte free cholesterol lipotoxicity results from JNK1-mediated mitochondrial injury and is HMGB1 and TLR4-dependent. J Hepatol. 2014;61:1376–1384.
87. Mari M, Caballero F, Colell A, et al. Mitochondrial free cholesterol loading sensitizes to TNF- and Fas-mediated steatohepatitis. Cell Metab. 2006;4:185–198.
88. Luukkonen PK, Sadevirta S, Zhou Y, et al. Saturated fat is more metabolically harmful for the human liver than unsaturated fat or simple sugars. Diabetes Care. 2018;41:1732–1739.
89. Pagadala M, Kasumov T, McCullough AJ, et al. Role of ceramides in nonalcoholic fatty liver disease. Trends Endocrinol Metab. 2012;23:365–371.
90. Xia JY, Holland WL, Kusminski CM, et al. Targeted induction of ceramide degradation leads to improved systemic metabolism and reduced hepatic Steatosis. Cell Metab. 2015;22:266–278.
91. Backhed F, Ley RE, Sonnenburg JL, et al. Host-bacterial mutualism in the human intestine. Science. 2005;307:1915–1920.
92. Cani PD. Human gut microbiome: hopes, threats and promises. Gut. 2018;67:1716–1725.
93. Flint HJ, Scott KP, Louis P, et al. The role of the gut microbiota in nutrition and health. Nat Rev Gastroenterol Hepatol. 2012;9:577–589.
94. Betrapally NS, Gillevet PM, Bajaj JS. Changes in the intestinal microbiome and alcoholic and nonalcoholic liver diseases: causes or effects? Gastroenterology. 2016;150:1745–1755.e1743.
95. Neish AS. Microbes in gastrointestinal health and disease. Gastroenterology. 2009;136:65–80.
96. Sommer F, Backhed F. The gut microbiota–masters of host development and physiology. Nat Rev Microbiol. 2013;11:227–238.
97. Turnbaugh PJ, Ley RE, Mahowald MA, et al. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature. 2006;444:1027–1031.
98. Leung C, Rivera L, Furness JB, et al. The role of the gut microbiota in NAFLD. Nat Rev Gastroenterol Hepatol. 2016;13:412–425.
99. den Besten G, van Eunen K, Groen AK, et al. The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism. J Lipid Res. 2013;54:2325–2340.
100. Cani PD, Bibiloni R, Knauf C, et al. Changes in gut microbiota control metabolic endotoxemia-induced inflammation in high-fat diet-induced obesity and diabetes in mice. Diabetes. 2008;57:1470–1481.
101. Giorgio V, Miele L, Principessa L, et al. Intestinal permeability is increased in children with non-alcoholic fatty liver disease, and correlates with liver disease severity. Dig Liver Dis. 2014;46:556–560.
102. Turner JR. Intestinal mucosal barrier function in health and disease. Nat Rev Immunol. 2009;9:799–809.
103. De Minicis S, Rychlicki C, Agostinelli L, et al. Dysbiosis contributes to fibrogenesis in the course of chronic liver injury in mice. Hepatology. 2014;59:1738–1749.
104. Miele L, Valenza V, La Torre G, et al. Increased intestinal permeability and tight junction alterations in nonalcoholic fatty liver disease. Hepatology. 2009;49:1877–1887.
105. Pierantonelli I, Rychlicki C, Agostinelli L, et al. Author correction: lack of NLRP3-inflammasome leads to gut-liver axis derangement, gut dysbiosis and a worsened phenotype in a mouse model of NAFLD. Sci Rep. 2017;7:17568.
106. Tomas J, Mulet C, Saffarian A, et al. High-fat diet modifies the PPAR-γ pathway leading to disruption of microbial and physiological ecosystem in murine small intestine. Proc Natl Acad Sci U S A. 2016;113:E5934–E5943.
107. Levy M, Kolodziejczyk AA, Thaiss CA, et al. Dysbiosis and the immune system. Nat Rev Immunol. 2017;17:219–232.
108. Miura K, Ohnishi H. Role of gut microbiota and toll-like receptors in nonalcoholic fatty liver disease. World J Gastroenterol. 2014;20:7381–7391.
109. Mridha AR, Haczeyni F, Yeh MM, et al. TLR9 is up-regulated in human and murine NASH: pivotal role in inflammatory recruitment and cell survival. Clin Sci. 2017;131:2145–2159.
110. Csak T, Velayudham A, Hritz I, et al. Deficiency in myeloid differentiation factor-2 and toll-like receptor 4 expression attenuates nonalcoholic steatohepatitis and fibrosis in mice. Am J Physiol Gastrointest Liver Physiol. 2011;300:G433–G441.
111. Miura K, Kodama Y, Inokuchi S, et al. Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1beta in mice. Gastroenterology. 2010;139:323–334.e327.
112. Miura K, Yang L, van Rooijen N, et al. Toll-like receptor 2 and palmitic acid cooperatively contribute to the development of nonalcoholic steatohepatitis through inflammasome activation in mice. Hepatology. 2013;57:577–589.
113. Himes RW, Smith CW. Tlr2 is critical for diet-induced metabolic syndrome in a murine model. FASEB J. 2010;24:731–739.
114. Cani PD, Amar J, Iglesias MA, et al. Metabolic endotoxemia initiates obesity and insulin resistance. Diabetes. 2007;56:1761–1772.
115. Spruss A, Kanuri G, Wagnerberger S, et al. Toll-like receptor 4 is involved in the development of fructose-induced hepatic steatosis in mice. Hepatology. 2009;50:1094–1104.
116. Rivera CA, Adegboyega P, van Rooijen N, et al. Toll-like receptor-4 signaling and Kupffer cells play pivotal roles in the pathogenesis of non-alcoholic steatohepatitis. J Hepatol. 2007;47:571–579.
117. Dapito DH, Mencin A, Gwak GY, et al. Promotion of hepatocellular carcinoma by the intestinal microbiota and TLR4. Cancer Cell. 2012;21:504–516.
118. Li J, Sung CY, Lee N, et al. Probiotics modulated gut microbiota suppresses hepatocellular carcinoma growth in mice. Proc Natl Acad Sci U S A. 2016;113:E1306–E1315.
119. Yoshimoto S, Loo TM, Atarashi K, et al. Obesity-induced gut microbial metabolite promotes liver cancer through senescence secretome. Nature. 2013;499:97–101.
120. Ponziani FR, Bhoori S, Castelli C, et al. Hepatocellular carcinoma is associated with gut microbiota profile and inflammation in non-alcoholic fatty liver disease. Hepatology. Published online April 17, 2018. DOI:10.1002/hep.30036.
121. Steib CJ, Hartmann AC, v Hesler C, et al. Intraperitoneal LPS amplifies portal hypertension in rat liver fibrosis. Lab Invest. 2010;90:1024–1032.
122. Lutz P, Parcina M, Bekeredjian-Ding I, et al. Impact of rifaximin on the frequency and characteristics of spontaneous bacterial peritonitis in patients with liver cirrhosis and ascites. PloS One. 2014;9:e93909.
123. Bass NM, Mullen KD, Sanyal A, et al. Rifaximin treatment in hepatic encephalopathy. N Engl J Med. 2010;362:1071–1081.
124. Boetticher NC, Peine CJ, Kwo P, et al. A randomized, double-blinded, placebo-controlled multicenter trial of etanercept in the treatment of alcoholic hepatitis. Gastroenterology. 2008;135:1953–1960.
125. Tilg H, Jalan R, Kaser A, et al. Anti-tumor necrosis factor-alpha monoclonal antibody therapy in severe alcoholic hepatitis. J Hepatol. 2003;38:419–425.
126. Verbeke L, Farre R, Verbinnen B, et al. The FXR agonist obeticholic acid prevents gut barrier dysfunction and bacterial translocation in cholestatic rats. Am J Pathol. 2015;185:409–419.
127. Ubeda M, Lario M, Munoz L, et al. Obeticholic acid reduces bacterial translocation and inhibits intestinal inflammation in cirrhotic rats. J Hepatol. 2016;64:1049–1057.
128. Neuschwander-Tetri BA, Loomba R, Sanyal AJ, et al. Farnesoid X nuclear receptor ligand obeticholic acid for non-cirrhotic, non-alcoholic steatohepatitis (FLINT): a multicentre, randomised, placebo-controlled trial. Lancet. 2015;385:956–965.
129. Arab JP, Karpen SJ, Dawson PA, et al. Bile acids and nonalcoholic fatty liver disease: molecular insights and therapeutic perspectives. Hepatology. 2017;65:350–362.
130. Zhou M, Yang H, Learned RM, et al. Non-cell-autonomous activation of IL-6/STAT3 signaling mediates FGF19-driven hepatocarcinogenesis. Nat Commun. 2017;8:15433.
131. Zhou M, Luo J, Chen M, et al. Mouse species-specific control of hepatocarcinogenesis and metabolism by FGF19/FGF15. J Hepatol. 2017;66:1182–1192.
132. Zhou M, Learned RM, Rossi SJ, et al. Engineered FGF19 eliminates bile acid toxicity and lipotoxicity leading to resolution of steatohepatitis and fibrosis in mice. Hepatol Commun. 2017;1:1024–1042.
133. Harrison SA, Rinella ME, Abdelmalek MF, et al. NGM282 for treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial. Lancet. 2018;391:1174–1185.
134. Vrieze A, Van Nood E, Holleman F, et al. Transfer of intestinal microbiota from lean donors increases insulin sensitivity in individuals with metabolic syndrome. Gastroenterology. 2012;143:913–916.e917.
135. Guo H, Callaway JB, Ting JP. Inflammasomes: mechanism of action, role in disease, and therapeutics. Nat Med. 2015;21:677–687.
136. Mridha AR, Wree A, Robertson AAB, et al. NLRP3 inflammasome blockade reduces liver inflammation and fibrosis in experimental NASH in mice. J Hepatol. 2017;66:1037–1046.
137. Henao-Mejia J, Elinav E, Jin C, et al. Inflammasome-mediated dysbiosis regulates progression of NAFLD and obesity. Nature. 2012;482:179–185.
138. Wree A, McGeough MD, Pena CA, et al. NLRP3 inflammasome activation is required for fibrosis development in NAFLD. J Mol Med. 2014;92:1069–1082.
139. Vandanmagsar B, Youm YH, Ravussin A, et al. The NLRP3 inflammasome instigates obesity-induced inflammation and insulin resistance. Nat Med. 2011;17:179–188.
140. Stienstra R, van Diepen JA, Tack CJ, et al. Inflammasome is a central player in the induction of obesity and insulin resistance. Proc Natl Acad Sci U S A. 2011;108:15324–15329.
141. Yeh MM, Brunt EM. Pathological features of fatty liver disease. Gastroenterology. 2014;147:754–764.
142. Gadd VL, Skoien R, Powell EE, et al. The portal inflammatory infiltrate and ductular reaction in human nonalcoholic fatty liver disease. Hepatology. 2014;59:1393–1405.
143. Sutti S, Jindal A, Locatelli I, et al. Adaptive immune responses triggered by oxidative stress contribute to hepatic inflammation in NASH. Hepatology. 2014;59:886–897.
144. Weston CJ, Shepherd EL, Claridge LC, et al. Vascular adhesion protein-1 promotes liver inflammation and drives hepatic fibrosis. J Clin Invest. 2015;125:501–520.
145. Wolf MJ, Adili A, Piotrowitz K, et al. Metabolic activation of intrahepatic CD8+ T cells and NKT cells causes nonalcoholic steatohepatitis and liver cancer via cross-talk with hepatocytes. Cancer Cell. 2014;26:549–564.
146. Inzaugarat ME, Ferreyra Solari NE, Billordo LA, et al. Altered phenotype and functionality of circulating immune cells characterize adult patients with nonalcoholic steatohepatitis. J Clin Immunol. 2011;31:1120–1130.
147. Ferreyra Solari NE, Inzaugarat ME, Baz P, et al. The role of innate cells is coupled to a Th1-polarized immune response in pediatric nonalcoholic steatohepatitis. J Clin Immunol. 2012;32:611–621.
148. Tang Y, Bian Z, Zhao L, et al. Interleukin-17 exacerbates hepatic steatosis and inflammation in non-alcoholic fatty liver disease. Clini Exp Immunol. 2011;166:281–290.
149. Sell H, Habich C, Eckel J. Adaptive immunity in obesity and insulin resistance. Nat Rev Endocrinol. 2012;8:709–716.
150. Ilan Y, Maron R, Tukpah AM, et al. Induction of regulatory T cells decreases adipose inflammation and alleviates insulin resistance in Ob/Ob mice. Proc Natl Acad Sci U S A. 2010;107:9765–9770.
151. Adar T, Ben Ya'acov A, Lalazar G, et al. Oral administration of immunoglobulin G-enhanced colostrum alleviates insulin resistance and liver injury and is associated with alterations in natural killer T cells. Clini Exp Immunol. 2012;167:252–260.
152. Elinav E, Pappo O, Sklair-Levy M, et al. Amelioration of non-alcoholic steatohepatitis and glucose intolerance in Ob/Ob mice by oral immune regulation towards liver-extracted proteins is associated with elevated intrahepatic NKT lymphocytes and serum IL-10 levels. J Pathol. 2006;208:74–81.
153. Lalazar G, Mizrahi M, Turgeman I, et al. Oral administration of OKT3 MAb to patients with NASH, promotes regulatory T-cell induction, and alleviates insulin resistance: results of a phase IIa blinded placebo-controlled trial. J Clin Immunol. 2015;35:399–407.
154. Friedman SL, Ratziu V, Harrison SA, et al. A randomized, placebo-controlled trial of cenicriviroc for treatment of nonalcoholic steatohepatitis with fibrosis. Hepatology. 2018;67:1754–1767.
155. Golabi P, Bush H, Stepanova M, et al. Liver transplantation (LT) for cryptogenic cirrhosis (CC) and nonalcoholic steatohepatitis (NASH) cirrhosis: data from the scientific registry of transplant recipients (SRTR): 1994 to 2016. Medicine (Baltimore). 2018;97:e11518.
156. Yalamanchili K, Saadeh S, Klintmalm GB, et al. Nonalcoholic fatty liver disease after liver transplantation for cryptogenic cirrhosis or nonalcoholic fatty liver disease. Liver Transpl. 2010;16:431–439.
157. Wang X, Li J, Riaz DR, et al. Outcomes of liver transplantation for nonalcoholic steatohepatitis: a systematic review and meta-analysis. Clin Gastroenterol Hepatol. 2014;12:394–402.e391.
158. Contos MJ, Cales W, Sterling RK, et al. Development of nonalcoholic fatty liver disease after orthotopic liver transplantation for cryptogenic cirrhosis. Liver Transpl. 2001;7:363–373.
159. Kemmer N, Neff GW, Franco E, et al. Nonalcoholic fatty liver disease epidemic and its implications for liver transplantation. Transplantation. 2013;96:860–862.
160. Kappus M, Abdelmalek M. De novo and recurrence of nonalcoholic Steatohepatitis after liver transplantation. Clin Liver Dis. 2017;21:321–335.
161. Mueller KM, Kornfeld JW, Friedbichler K, et al. Impairment of hepatic growth hormone and glucocorticoid receptor signaling causes steatosis and hepatocellular carcinoma in mice. Hepatology. 2011;54:1398–1409.
162. Dolinsky VW, Douglas DN, Lehner R, et al. Regulation of the enzymes of hepatic microsomal triacylglycerol lipolysis and re-esterification by the glucocorticoid dexamethasone. Biochem J. 2004;378:967–974.
163. Orlando G, Baiocchi L, Cardillo A, et al. Switch to 1.5 grams MMF monotherapy for CNI-related toxicity in liver transplantation is safe and improves renal function, dyslipidemia, and hypertension. Liver Transpl. 2007;13:46–54.
164. Lopes PC, Fuhrmann A, Sereno J, et al. Short and long term in vivo effects of cyclosporine A and sirolimus on genes and proteins involved in lipid metabolism in Wistar rats. Metabolism. 2014;63:702–715.
165. Fuhrmann A, Lopes \, Sereno J, et al. Molecular mechanisms underlying the effects of cyclosporin a and sirolimus on glucose and lipid metabolism in liver, skeletal muscle and adipose tissue in an in vivo rat model. Biochem Pharmacol. 2014;88:216–228.
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