FOCUS ON NAFLD

From NAFLD to NASH to cirrhosis

new insights into disease mechanisms
Alexander Wree, Lori Broderick, Ali Canbay, Hal M. Hoffman and Ariel E. Feldstein
Abstract | NAFLD has evolved as a serious public health problem in the USA and around the world. In fact,
NASHthe most serious form of NAFLDis predicted to become the leading cause of liver transplantation
inthe USA by the year 2020. The pathogenesis of NAFLD and NASH, in particular the mechanisms responsible
for liver injury and fibrosis, is the result of a complex interplay between host and environmental factors, and is
at the centre of intense investigation. In this Review, we focus on recently uncovered aspects of the genetic,
biochemical, immunological and molecular events that are responsible for the development and progression
of this highly prevalent and potentially serious disease. These studies bring new insight into this complex
disorder and have led to the development of novel therapeutic and diagnostic strategies that might enable
apersonalized approach in the management of this disease.
Wree, A. et al. Nat. Rev. Gastroenterol. Hepatol. 10, 627636 (2013); published online 20 August 2013; doi:10.1038/nrgastro.2013.149

Introduction
NAFLD has become the most common cause of chronic
liver disease. Estimates of NAFLD prevalence based on
cryptogenic abnormal liver function, autopsy samples,
and findings from ultrasonography and magnetic resonance spectroscopy are 337%, with the usual figure
quoted at ~30% (reviewed elsewhere1,2). The spectrum of
NAFLD ranges from simple steatosis to NASH to fibrosis
and cirrhosis. NASH, characterized by hepatocellular lipid
accumulation (steatosis) along with inflammation and
varying degrees of fibrosis,3 is a potentially serious condition, and ~1025% of patients with NASH might progress
to cirrhosis.4,5 Moreover, NAFLD can progress to hepatocellular carcinoma in the absence of apparent cirrhosis.6
However, the general prevalence of NASH is difficult to
determine, as invasive liver biopsy remains the diagnostic
gold-standard approach. A review of the epidemiology of
NAFLD found that in studies of patients who had undergone bariatric surgery (16 individual studies, including
2,956 patients), 76% had steatosis (range reported in the
studies 3399%), 37% had NASH (9.872.5%), 23% had
fibrosis (7.349%) and 5.8% had cirrhosis (1.610%).7 As
NAFLD is becoming increasingly common in the developed world, NASH is projected to become theleading
cause of liver transplantation in the USA by 2020.8
The clinical importance of NAFLD and the current
lack of effective medications to stop or reverse disease
progression in patients with NASH have sparked great
interest and intense investigation into the basic mechanisms involved in the development and progression of
the disease. The current, and most accepted, concept out
lining the pathogenesis of NAFLD involves multiple hits.9
These hits are characterized by the occurrence of parallel
Competing interests
The authors declare no competing interests.

events that involve a complex interaction and crosstalk

between environmental factors, host genetics and gut
microflora.10,11 This interaction might promote isolated
steatosis, innate immune activation, inammation, cell
death or progressive liver damage.9
In this Review, we present new insights into NAFLD
and suggest novel therapeutic and diagnostic strat
egies that might enable a personalized approach to
managing the disease. We integrate the role and influence of dietary factors, focusing on certain lipid and
carbohydrate components; the interplay between host
genetics and environment; the role of innate immune activation andinflammation; and the effect of hepatocellular
injuryand cell death in the progression of NAFLD.

Dietary factors and NAFLD progression

Consumption of high-caloric diets rich in lipids result
in weight gain, obesity and insulin resistancewellestablished risk factors for the initial events leading to the
development of fatty liver. Further evidence now suggests
that the composition of the diet, in particular the types of
lipids and carbohydrates, also have an important role in
the progression of disease to NASH and fibrosis (Figure1).

Dietary lipids: PUFAs

Polyunsaturated fatty acids (PUFAs) can have a pro
inflammatory or anti-inflammatory effect depending on
their structure and have been shown to have an important role in the development of NAFLD. n6 PUFAs, such
as linoleic acid and arachidonic acid, are precursors to
potent proinflammatory eicosanoids.12,13 By contrast,
n3 PUFAs, such as linolenic acid, eicosapentaenoic
acid and do cosahexaenoic acid, have an important
anti-inflammatory role; they decrease lipogenesis and
increase fatty acid oxidation, leading to a reduction in

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Department of
Pediatrics, University
ofCalifornia, San
Diego, 9500 Gilman
Drive, La Jolla,
CA920370715, USA
(A. Wree, L.Broderick,
H.M.Hoffman,
A.E.Feldstein).
UniversityHospital
Essen, Department
ofGastroenterology
and Hepatology,
Hufelandstrasse 55,
45122 Essen, Germany
(A. Canbay).
Correspondence to:
A.E. Feldstein
afeldstein@ucsd.edu

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REVIEWS
Key points
The composition of the diet, in particular the types of lipids (mainly omega6
fatty acids) and carbohydrates (mainly fructose), have an important role in the
progression of NAFLD to NASH and fibrosis
A complex interplay between the environment (especially diet), host genetics
and the gut microflora is crucial for the development and progression of NAFLD
Activation of the innate immune system has an essential role in maintaining
homeostasis and liver regeneration, as well as disease pathogenesis, acting
ina cooperative rather than independent fashion
Discoveries that characterized the importance of cell death in NAFLD
progression triggered the development of novel therapeutic and diagnostic
approaches for NAFLD
Various types of cell death contribute to the development of NAFLD; extensive
crosstalk and biochemical cooperation exists between these cell death
pathways to drive disease progression

hepatic steatosis.1416 Patients with NAFLD have been

shown to have lower fish and n3 PUFA consumption
than those without fatty liver.1719 Moreover, lipidomic
studies in patients with NAFLD or NASH have consistently demonstrated an association between a high
n6:n3 ratio in both blood and liver, with the presence
and severity of NAFLD.20,21 Manipulating the diet to
alter this ratio seems to be an attractive and safe target
for the treatment of NASH. Most studies thus far have
taken theapproach ofsupplementing patients with n3
PUFAs. A meta-analysis in 2012 of nine studies including a total of 355 patients with NAFLD suggested that
n3 PUFA supplementation had a beneficial effect on the
levels of liver fat and liver enzymes.19 However, substantial
statistical heterogeneity existed between the studies, and
trial design varied markedly, indicating the need for large
well-designed, randomized controlled trials.
A second approach to improve the n6:n3 ratio involves
decreasing the consumption of n6 PUFAs. Indeed, the
Liver

Inflammation

Hepatocyte

intake of linoleic acidthe most abundant n6 PUFA in

human dietsis ten times higher in the USA when compared to the Philippines and doubled when compared to
European countries.22 Linoleic acid is a major component
of human tissues and can form bioactive oxidized linoleic
acid metabolites (OXLAMs), either enzymatically (primarily via 12/15-lipoxygenase), or nonenzymatically by freeradical-mediated oxidation in response to oxidative stress.23
Levels of specific OXLAMs are selectively increased in
blood in both adults and children with NASH and correlate
with the level of liver damage in these patients.23 Moreover,
levels of circulating OXLAMs seem to be dynamic and they
fluctuate in concert with changes in histological features
of disease severity over time.24 Experimental studies using
12/15-lipooxygenase knockout mice showed a reduction
in hepatic inflammation and lymphocyte homing, which
are normally induced by a high-fat diet with a high n6:n3
ratio.25 Moreover, feeding rats peroxidized fatty acids
directly triggered hepatic inflammation, with increased
hepatic production of Il1 and Tnf.26 These findings in
rodents can be translated to humans: a pilot study in 2012
demonstrated that a 12-week dietary intervention to reduce
linoleic acid levels in patients with chronic headaches
markedly reduced the abundance of plasma OXLAMs.27
The linoleic acid content of multiple circulating lipid fractions, which could act as precursor pools for endogenous
OXLAM synthesis, was also reduced.27 Future studies
targeted at lowering the dietary intake of linoleic acid in
patients with NAFLD while monitoring blood OXLAM
levels and liver h
istology are warranted.

Dietary carbohydrates: fructose

Excess carbohydrate consumption has been extensively
linked with the development of NAFLD.28 Growing attention has focused on fructose, a highly lipogenic sugar that
Gastrointestinal
tract

Steatosis

CH3
-Linoleic acid (n-3)
O
OH
Fructose
CH2OH

HO
Mitochondria

HO

CH2OH

OH

Linoleic acid (n-6)

Gut
microbiome

O
OH

Beneficial effect
Deleterious effect
Various effects

Figure 1 | Influence of dietary factors and genetics in development of NAFLD. PUFAs have been linked to the development
of liver steatosis and liver inflammation. They have differential effects on liver inflammation: n3 PUFAs (-linolenic acid)
have anti-inflammatory effects, whereas n6 PUFAs (linoleic acid) serve as precursors for potent inflammatory eicosanoids.
The most common component of all major caloric sweeteners, fructose, is highly lipogenic. The interplay of host genetics
and environment, such as the gut microbiota, has been shown to determine the disease phenotype and the progression
tothe more advanced forms of the disease. Abbreviation: PUFA, polyunsaturated fatty acid.

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FOCUS ON NAFLD
is a common component in all major caloric sweeteners
(sucrose, high-fructose corn syrup, honey and fruit juice
concentrates).29 Indeed, a growing number of epidemiological and experimental studies have supported a potentially causative role for increased consumption of this
sugar with increasing rates of obesity and its metabolic
complications, including NAFLD.3033
Studies in different animal models have examined the
mechanisms by which fructose induces NAFLD. C57BL/6
mice fed a high-caloric, 65% sucrose diet for 8weeks
exhibited obesity, insulin resistance and macrovesicular
steatosis.34 The mechanisms proposed by the authors of
the study include increased hepatic denovo lipogenesis, as
well as activation of inflammatory pathways. In humans,
excessive fructose intake has been associated with
increased levels of plasma triglycerides and hepatic lipid
deposition owing, in part, to increased hepatic denovo
lipogenesis.35 Patients with NAFLD were also shown to
display increased soft drink consumption independent
of metabolic syndrome diagnosis and soft drink consumption was a strong predictor of fatty liver (OR 2.0,
P<0.04).36 In a study conducted by the NASH Clinical
Research Network, food questionnaire data were collected from 427 adults within 3months of a liver biopsy;
fructose consumption was associated with a reduced
steatosis grade, but increased fibrosis stage (P<0.05 for
each).37 However, the mechanisms responsible for these
associations were not further explored.
Of note, a singular role for a high-fructose diet in the
pathogenesis of NAFLD in humans has been questioned
by evidence that fructose intake at normal population
levels and patterns does not cause substantially different
biochemical outcomes when compared with consumption of other dietary sugars, such as glucose.38 Moreover,
limited data suggest that consumption of free fructose
(as provided in high-fructose corn syrup) causes more
adverse effects in the liver than consumption of fructose
consumed in combination with sucrose.39 Furthermore,
experimental models that use supraphysiological levels of
fructose in isolation, or substantially alter the usual dietary
glucose:fructose ratio, might not be predictive of typical
human outcomes.38 Future studies, including prospective
controlled trials with liver histological endpoints, are
needed to define the amount of fructose that is safe for
patients with NAFLD to consume and to determine the
extent and mechanisms by which fructose contributes to
the pathogenesis and progression of thisdisease.

Interplay of host genetics and environment

Growing evidence supports the concept that NAFLD is a
highly heritable disease in which genetic variations and
environment closely interact to determine the disease
phenotype and progression to the more advanced forms
of the disease. Human genome-wide association studies
(GWAS) have provided important insights into the
genomic variation in NAFLD and have identified specific
loci that contribute to fat accumulation in the liver and
NASH development (Supplementary Table1).40 A major
limitation of most of the GWAS, as well as other genetic
studies in NAFLD, has been the lack of assessment of the

crosstalk between genetic variations with key environ

mental factors that influence the development of NAFLD
and progression to NASH. Parks and colleagues used
a system genetics approach to shed light on the crucial
interplay between genetics, diet and gut microbiota in
the control of obesity in mice.41 A high-calorie, high-fat
and high-carbohydrate diet induced different degrees of
obesity in >100 inbred strains of mice; these differences
were strain dependent and were not accounted for by
food intake.41 The analyses identified 11 genome-wide
loci associated with obesity traits in these mice, several of
which overlap with loci previously identified in humans.
The high-caloric diet also induced marked changes in
gut microbiota composition, which was strongly influenced by the genetic background of the mouse (that is,
some strains showed large shifts in gut flora composition
whereas others did not). The genetic predisposition
of 129S6 mice to impaired glucose homeostasis and
NAFLD was shown to be associated with disruptions
in choline metabolism, characterized by low levels of
plasma phosphatidylcholine and high urinary excretion
of methylamines.42 As cholineis converted into methyl
amines by gut microbiota in these mice on a high-fat diet,
the bioavailability of choline is reduced, mimicking the
effect of choline-deficient diets and leading to NAFLD.
Two studies have provided evidence for the interplay between host genetics and environment in human
NAFLD.43,44 In the first study, a diet with a high n6:n3
PUFA ratio resulted in increased hepatic fat fractions in
a large cohort of children and adolescents (n=85) from
an obesity clinic.43 However, this effect was seen only in
children with a genetic polymorphism in patatin-like
phospholipase domain containing3 (PNPLA3), which
has been identified as the strongest modifier of NAFLD
and NASH pathogenesis in most of the GWAS performed
to date. The second study demonstrated that the genetic
association between hepatic fat accumulation and liver
cell death might be, in part, dependent on ethnicity. 44
Indeed, in >220 children with obesity, the same degree of
hepatic fat content in African-American youths was associated with lower levels of liver cell death than white and
Hispanic youths, which was independent of the degree of
insulin resistance.44 Future studies taking a system biology
approach and integrating data from novel gene technol
ogies (such as whole-genome or whole-exome sequencing and transcriptome sequencing) with environmental
factors, metagenomics and epigenomics are warranted and
will result in a marked advancement in our understanding
of complex metabolic disorders such as NAFLD.

Innate immunity and inflammation

Innate immunity is generated through the integration
of germline-encoded mechanisms of pattern recognition, leading to cellular and humoral (complement)
responses to foreign and endogenous danger signals.
These p
athogen-associated molecular patterns (PAMPs)
and damage-associated molecular patterns (DAMPs) are
recognized by four classes of pattern-recognition receptors (PRR): Toll-like receptors (TLRs), nucleotide-binding
oligomerization domain (NOD), leucine-rich-repeat

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REVIEWS
containing receptors (NLRs), and others (reviewed elsewhere45,46). In the liver, activation of the innate immune
system has a critical role in maintaining homeostasis and
liver regeneration, as well as disease pathogenesis, acting in
a cooperative rather than independent fashion. Although
the role of innate immune components such as mast cells,
natural killer cells, complement pathway proteins, secreted
PRRs, and acute phase reactants have been implicated in
liver disease (reviewed in47), for this Review, we will focus
on recently reported aspects of TLRs, NLR inflammasomes
and natural killer Tcells (NKT cells) in NASH.

Toll-like receptors
TLRs comprise a family of cell surface and endocytic
receptors that recognize specific invariant motifs on
pathogens, including peptidoglycan (TLR2), lipopolysaccharide (LPS; TLR4), DNA viruses and unmethylated
CpG motifs (TLR9).45,46 TLRs are expressed at low levels
throughout the liver including Kupffer cells, hepatic stellate cells (HSCs), biliary epithelial cells, sinusoidal cells
and dendritic cells.4851 Aberrations in TLR signalling have
been implicated in multiple liver diseases including liver
fibrosis, ischaemiareperfusion injury, viral hepatitis and
hepatocellular carcinoma, probably by elevating exposure to PAMPs and promoting an inflammatory tissue
microenvironment. Discussion of TLR signalling in the
pathogenesis of these diseases has been reviewed.51,52 For
NASH, the vast majority of work has traditionally focused
on TLR4 and TLR9, and more recently on TLR2.
TLR4
TLR4 is most widely recognized for its activation in
response to LPS. Mouse models have shown that NASH
induced by high-fat, high-fructose and methionine
choline deficient (MCD) diets leads to enhanced expression of Tlr4, as well as its co-receptors, with measurable
portal endotoxemia.53 Wild-type mice on standard diets
receiving low-dose infusions of LPS also develop a steato
hepatic phenotype, emphasizing the importance of the
gutliver axis in disease pathogenesis. 53 By contrast,
Tlr4 mutant mice display reduced levels of diet-induced
steatosis, indicating that inflammation and fibrogenesis
are dependent on Tlr4-mediated signalling in Kupffer
cells and HSCs.54 Further work on the precise signalling
mechanisms has suggested that downstream chaperone
proteins, transcription factors and reactive oxygen species
(ROS) might differentially affect progression of NASH.55
These mouse models suggest a multiple hit model
leading from benign steatosis to steatohepatitis, consistent withthe theories of disease development in patients
with NAFLD.56 Given the role of TLRs in host protection,
targeted manipulation of these pathways might lead to
promising treatment strategies for NASH.
TLR9
Endosomal TLR9 recognizes unmethylated CpG motifs,
which are prevalent in bacterial DNA and endogenous
ligands from apoptotic hepatocyte DNA. Human and
mouse HSCs treated with CpG or apoptotic hepatocyte
DNA led to increases in levels of fibrotic markers and
630 | NOVEMBER 2013 | VOLUME 10

morphological evidence of cellular activation that could

be blocked by TLR9 antagonists.57,58 Invivo models of
hepatic fibrosis further support a role for TLR9 in HSC
activation and fibrosis, but a direct role for TLR9 in
NAFLD-induced fibrosis has yet to be defined. Invivo
and invitro studies demonstrate that TLR9MyD88 signalling also mediates IL1 production in Kupffer cells,
which stimulates both hepatocytes and HSCs, leading to
progression of NASH.59 Indeed, blockage of Il1 signalling
in mice led to a reduction of Tlr9-mediated liver injury,
and disruption of this pathway might be an important
therapeutic opportunity.59 IL1 is a key downstream
mediator in NASH progression and will be discussed
further in the section on inflammasomes.
TLR2
TLR2 recognizes a wide variety of ligands, including
bacterial, fungal and protozoal components and has the
unique ability to discriminate among specific ligands.
TLR2 activation is associated with production of an array
of proinflammatory cytokines and chemokines. Given
this complexity, it is not surprising that our understanding of the role of TLR2 in NAFLD is in its early stages,
and results to date have been conflicting. Initial studies
demonstrated that the absence of Tlr2 signalling was protective in mouse models of steatosis induced by a high-fat
diet.60 In 2013, Miura etal.61 extended these early findings
and showed that Tlr2 knockout mice have less inflam
mation after feeding with a choline-deficient amino-aciddefined diet known to induce NASH. Other investigators,
however, demonstrated that Tlr2 knockout mice given a
MCD diet had increased Tlr4 expression and an enhancement of NASH.62 Interestingly, the protective role of Tlr2
was negatively influenced by the presence of dietary saturated fat. Although additional studies are needed, these
contradictory findings emphasize the variable response
of TLR2 to exogenous stimuli. For patients with NAFLD,
they further underscore a role for diet in modulation of
innate immunity and how manipulation of diet might
provide a therapeutic benefit.

NLR inflammasomes
NLRs intersect with a wide variety of immune and cell
death pathways. A few NLRs, of which the most wellstudied is NLRP3, form cytoplasmic multiprotein complexes known as inflammasomes, which are comprised
of adaptor proteins such as the apoptosis-associated
speck-like protein containing a CARD (ASC, encoded by
PYCARD) and the serine protease caspase1 (Figure2).
Caspase1 has a role in cell death (discussed later),
andis also the primary mechanism for the cleavage and
release of IL1 and IL18.63 Inflammasomes, (in particular NLRP3 activation) and IL1 signalling, have been
implicated in the pathogenesis of many liver diseases
including ischaemiareperfusion injury, drug-mediated,
pathogen-mediated or endotoxinmediated pathology, as
well as NAFLD and liver fibrosis.64
The effect of diet and gut microbiota on activating
inflammasome pathways in the liver has been the subject
of intense investigation.65 Given the role of the liver as a

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FOCUS ON NAFLD
first-pass organ in detoxification and interaction with the
gut, the inflammasome in the liver probably has a critical role in maintaining homeostasis between host microbial flora and regulation of inflammation. Moreover, the
inflammasome is known to be activated by ROS, and
ROS are suggested to be involved in TLR4-mediated
inflammation in in vivo models of NASH.55,56
Downstream of inflammasome activation, the importance of IL1 signalling in NASH has been demonstrated
in numerous mouse models of diet-induced NASH,
using various knockouts in the Il1 pathway. The pathological features of NASH are reduced in these mice, but
increased in mice deficient for the natural inhibitor of IL1
signalling, Il1 receptor antagonist (Il-1ra).66,67 A similar
reduction of diet-induced NASH in caspase1-deficient
mice was reported by Feldstein and colleagues, which is
consistent with these previous reports.68 This paradigm
is supported by the presence of increased IL1Ra levels in
serum and increased ILR hepatic expression in patients
with NASH.69 These studies suggest that biologic targeting
of IL1 signalling is a promising therapeutic strategy.
Studies with Nlrp3 knockout mice, however, have produced conflicting results. NLRP3 is clearly important in
fatty acid-induced inflammation through a variety of mechanisms, and Nlrp3 knockout mice fed a high-fat diet for
9months (obese) demonstrated considerably reduced pathological liver changes.70,71 However, a study in 2012 revealed
a more complex role for NLRP3, involving interaction with
gut microbiota.72 Using a 4-week mouse model (nonobese)
of NAFLD and NASH, knockouts for NLRP3 inflammasome components (Nlrp3, Pycard, and Casp1) as well as
Il18 showed modestly increased levels of steatosis and
inflammation in the liver. Interestingly, this phenotype was
transmissible to wild-type mice that were co-housed with
knockout mice, suggesting that inflammasome-deficient
mice have an altered communicable gut microbiota that
drives a heightened diet-induced liver disease. Therefore,
liver inflammasomes probably have a crucial role in maintaining homeostasis between host gut microbial flora and
regulation of inflammation associated with NASH.

Natural killer Tcells

Advanced NASH has an increased inflammatory cell infiltrate in the liver compared with steatosis, suggesting that
disease progression is driven by inflammation. The healthy
liver has NKT cells present in the hepatic sinusoids, which
are believed to act as essential links between innate and
adaptive immunity with roles in viral infections, tumour
immunity and autoimmune diseases. Their precise role
in NAFLD remains controversial and highlights gaps in
our knowledge of NKT cells, as well as in the progression
from steatosis to NAFLD. In addition, there are notable disparities in natural killer, NKT and invariant NKT (CD1ddependent, iNKT) cell populations between human and
mouse livers, underscoring the difficulties in identifying
their role in NAFLD.73 Thus, the contribution of different
NKT-cell subsets in disease progression remains unclear.
In humans, changes in the frequency of peripheral and
parenchymal NKT cells have been correlated with NAFLD
progression. Steatosis is associated with decreased numbers

Signal 2

DAMPs

PAMPs

Signal 1

TLR2

IL-1Ra
TLR4
TLR9

NLRP3
Signal 1

IL-1R

Nucleus
IL-1

ASC
Pro-caspase-1

Signal 1
Pro-IL-1

Active
caspase-1

Pro-IL-18
Signal 2
IL-18
NLRP3
inflammasome

Figure 2 | Mechanisms of NLRP3 inflammasome activation. Inflammasome

components such as NLRP3, NLRP1, ASC and caspase1 are expressed in Kupffer
cells and hepatocytes. Inflammasome activation is dependent on two successive
signals. Signal1 (dotted lines), driven by TLR and IL1R signalling, includes
expression of component proteins including NLRP3, ASC and pro-caspase1 and
pro-IL1 and pro-IL18.117 Signal2 is provided by PAMPs and/or DAMPs leading
tooligomerization of the inflammasome components and cleavage of caspase1,
which ultimately leads to release of active proinflammatory cytokines. IL1Ra
inhibits downstream effects of IL1, as well as blocking the IL1-driven
autoinflammatory loop. Abbreviations: DAMP, damage-associated molecular
pattern; PAMP, pathogen-associated molecular pattern; TLR, Toll-like receptor.

of liver NKT cells and immunohistochemical evaluation of

human NASH biopsy sections showed decreasing numbers
of NKT cells as steatosis grade increased.74 By contrast,
patients with NASH have livers enriched with NKT cells.75
Furthermore, the number of NKT cells and their cytokine
production correlated with NAFLD activity scores, indicating a role in disease enhancement. Similarly, Adler and
colleagues, in the largest patient study to date, showed that
NKT cells are increased in the liver and blood of patients
with moderate-to-severe steatosis, whereas there is no
marked difference in iNKT cells.76
Mouse models of fibrotic liver disease have supported
the findings in patients. These models have focused largely
on iNKT cells, although results from different models have
been conflicting. Mice lacking NKT cells are protected
from diet-induced fibrosis, possibly owing to decreased
activation of Hedgehog and osteopontin pathways; both
of these pathways have been shown to correlate with
disease progression in human NASH.77 Similarly, mice in
which NKT-cell retention and viability are increased have
enhanced NASH progression by promoting NKT-cellmediated responses.78,79 By contrast, other investigators
have demonstrated that NKT-cell depletion led to metabolic changes observed in steatosis which, similar to TLR
responses, might be influenced by diet.80,81 These findings

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REVIEWS
Autophagy

Apoptosis

Necroptosis

Nuclear
fragmentation

Plasma membrane
permeabilization
Membrane
blebbing

Autophagosome

mTOR
tBID

BCL2

CASP-8,10

Increased
cellular
volume

CASP-8

RIP-1

RIP-1 RIP-3

RIP-3

Pro-survival

RIP-1 RIP-3
Receptor
(e.g. TNFR1)

Receptor
(e.g. TNFR1)

Receptor
(e.g. growth factor)

Necrosis/oncosis

Pyroptosis

Nuclear
fragmentation

RIP-1 RIP-3
Swelling of
organelles

Cell
shrinkage
CASP-3,6,7

Vacuolization
of cytoplasm

Necroptosome

Apoptosome

Beclin 1

Release of
cellular
contents

Release of cellular
contents

Release of cellular
contents
Pore
formation
IL-1

IL-18

Cytosolic
swelling

CASP-1
Ipaf

AIM2
NLRP1

Release of
necrotic
blebbs

Mild DNA
damage

ATP
levels

Increased
cellular
volume

NLRP3

Flagellin

dsDNA
Toxins

DAMPs

Swelling of
organelles

ROS

PAMPs

Receptor
e.g. TLR4

Figure 3 | Hepatocellular injury and cell death. Forms of programmed cell death, such as autophagy, apoptosis, necroptosis,
andpyroptosis, have distinct signalling pathways and specific initiating events. The terminal event can be either lytic
(necroptosis, pyroptosis and necrosis), accompanied by cellular swelling and release of proinflammatory cellular contents,
ornonlytic (autophagy and apoptosis), which is associated with the breakdown of cellular components and intact plasma
membranes. However, there is frequent overlap and crosstalk between different types of cell death. The formation of the
autophagosome depends on the formation of a complex incorporating Beclin1, and is regulated by mTOR as well asby the antiapoptotic protein BCL2. BCL2 links the regulation of autophagy to apoptosis, which can be initiated via an intrinsic or extrinsic
pathway. The extrinsic pathway is based on activation of cell death receptors which trigger formation of the death complex,
activation of initiator caspases followed by effector caspases and the proapoptotic protein BID. tBID enhances the apoptotic
signal via crosstalk with the intrinsic (mitochondrial) pathway, generating more cleaved effector caspases which ultimately form
the apoptosome. Abbreviations: DAMP, damage-associated molecular pattern; PAMP, pathogen-associated molecular pattern;
ROS, reactive oxygen species; tBID, truncated BID.

are consistent with previous work that high-fat diets alter

the availability of liver ceramides, a primary antigen for
iNKT cells. The lack of stimulation by liver ceramides led
to depletion of hepatic iNKT cells and hepatic steatosis,
but a similar response has yet to be studied in patients
with NAFLD. 80 Similarly, iNKT knockout mice had
enhanced diet-induced liver inflammation characterized
by enhanced gene expression of inflammatory cytokines,
chemokines and by T-cell accumulation in the liver. 81
These studies suggest an immunoregulatory function of
iNKT cells in suppressing liver inflammation and contributing to maintenance of homeostasis in the liver. Clearly,
further investigation is necessary.
632 | NOVEMBER 2013 | VOLUME 10

Hepatocellular injury and cell death

Hepatocyte cell death and the intrinsic molecular signalling events leading to death have been shown to be
central to the development of NAFLD in both animal
models and humans (Figure3). Traditionally, hepatocyte cell death was morphologically bifurcated into two
mutually exclusive formsprogrammed (apoptotic cell
death) and accidental (necrotic cell death). New breakthroughs, however, have identified extensive crosstalk
and biochemical cooperation in the execution of different types of cell death. Table1 summarizes the character
istics of the main pathways. Typically, cell death has a
specific initiating cellular event, distinct signalling

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FOCUS ON NAFLD
Table1 | Forms of programmed and nonprogrammed cell death
Cell death pathway

Signalling

Lytic

Inflammation

Pathways

Apoptosis

Programmed

No

No

Intrinsic pathway: mitochondrial dysfunction

Extrinsic pathway: death receptor
Both pathways: caspases 3, 6 and 7

Autophagy

Programmed

No

No

Beclin 1
Autophagosome

Necroptosis

Programmed

Yes

Yes

Death receptor RIP-1 and RIP-3

Pyroptosis

Programmed

Yes

Yes

Caspase-1 activation

Oncosis or necrosis

Accidental

Yes

Yes

Mitochondrial dysfunction reactive oxygen species

transduction pathways and specific terminal cellular

events. Terminal events exhibit either lytic release of
cellular components (necrosis and pyroptosis) or breakdown of cellular components with intact plasma membranes (apoptotic and autophagic cell death) (Figure3).
However, there is frequent overlap and crosstalk between
modes.82 Background information for the cell death types
are given in Supplementary Box1 online.

Apoptosis
Apoptosis, a highly organized and genetically controlled
process, is the most investigated and best-defined form
of programmed cell death. Apoptotic cell death, mainly
in hepatocytes, has been linked with lipotoxic events and
fibrogenesis in NAFLD and NASH.
In hepatocytes, certain lipids, such as free fatty acids
(FFAs), can induce lysosomal permeablization and mitochondrial dysfunction (commonly referred to as lipotoxicity), which lead to apoptosis.8386 Notably, mice that
are genetically deficient for cathepsinB, and therefore
unable to induce lysosomal-driven apoptosis, are protected against diet-induced steatosis and liver injury.8789
In addition, FFAs have been shown to trigger the intrinsic apoptotic pathway, either directly or by increasing
proteasome-dependent loss of the key anti-apoptotic
protein MCL1.90,91
Fibrosis is based on activation of HSCs and experimental studies suggest that hepatocyte apoptosis and
the resulting apoptotic bodies are important activators
of HSCs.92 Indeed, apoptotic bodies from hepatocytes
are engulfed by HSCs, stimulating the fibrogenetic activity of these cells93 and DNA fragments from apoptotic
hepatocytes can also activate HSCs.57 Notably, attenuation of hepatocyte apoptosis reduces fibrogenesis in
animal models of cholestasis, whereas mice lacking the
anti-apoptotic Bcl-xL protein show marked increases in
hepatocyte apoptosis and liverfibrogenesis.9497
The increased levels of hepatocyte apoptosis in
patients with NASH has been linked to several receptors, which activate the extrinsic apoptotic pathway.
For example, Fas protein expression was higher in
patients with NASH than in patients with steatosis.98
An increase in TNF and TNF-1R expression has also
been reported in the liver of patients with NASH, which
correlated with the stage of liver fibrosis.99 Lysosomal
destabilization by FFAs resulted in activation and release
of cathepsinB into the cytosol, and stimulated TNF

expression, linking the intrinsic and extrinsic pathway

in NASHdevelopment.87

Autophagy and autophagic cell death

Autophagy is a conserved phenomenon that controls
important homeostatic functions, namely protein
degradation and organelle turnover.100 Cellular stress,
such as nutrient or growth factor deprivation, induces
rapid upregulation of autophagy to provide alternative sources for energy generation, thereby enabling
continuous cellsurvival.
Autophagy is now linked with several events in the
development of NAFLD. Decreased autophagic function drives the initial development of hepatic steatosis
and its progression to liver injury. Indeed, inhibition of
autophagy in cultured hepatocytes leads to markedly
increased levels of cellular triglycerides after they are challenged with methionine-deficient and choline-deficient
medium.101 In addition, autophagy might protect against
cell death, including the removal of damaged organelles
or proteins that contribute to cellular dysfunction. 102
The removal of mitochondria, socalled mitophagy, is
especially important and has been observed in hepatocytes and livers and could, t herefore, be be neficial
inNASH.103
Autophagy might also have an effect on the activation
of HSCs. Quiescent HSCs store lipids primarily in the
form of vitaminA and these lipids are lost during activation and transdifferentiation to myofibroblasts.104 It is
unclear how this process is mediated and two mechanisms that involve autophagy have been proposed. The
first postulates that an increased rate of autophagy is
required for lipid removal to provide energy to enable
activation. The second emphasizes the role of autophagy
in transdifferentiation of cells, as studies showed that
altered macroautophagy inhibits differentiation between
white and brown adipocytes.105 Additional studies are
clearly required to fully understand these mechanisms
and the role of autophagy in NAFLD.
Necroptosis
The term necroptosis has been introduced to describe a
receptor-induced, caspase-independent, cell death. It is
a highly regulated type of programmed cell death with a
morphological resemblance to necrosis.106 The contribution of necroptosis to liver pathology has been addressed
in several studies, but is still only partially understood.

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REVIEWS
Cytochrome (CYP2E1)-dependent receptor-interacting
protein (RIP)-3 expression induces hepatocyte necrop
tosis during ethanol feeding and ethanol-inducedhepatocyte death is RIP-3-dependent.107 In addition, hepatocytes
are capable of fulfilling a TRAIL-induced necroptosis, which is dependent on RIP-1RIP-3 complex
formation. Indeed, administration of a RIP-1 pharmacological inhibitor (necrostatin1) protected against concavalinAinduced hepatitis in mice.108 Liedtke etal.109
addressed the differential effects of TNF-induced liver
injury in hepatocyte-specific caspase8 knockout mice.
Those mice were protected, as expected, from liver failure
induced by Fas and Tnfr1, but injection of concavalinA resulted in marked oxidative damage and necrotic
injury.109 Compared with Fas-induced liver injury, concavalinA is mediated by membrane-bound TNF and
does not require suppression of NFB. Furthermore,
concavalinA is not caspase-dependent, which points to
necroptosis as the cause ofliver cell death. Future studies
addressing the role of necroptosis in NASH, and the
potential crosstalk with other modalities of cell death,
are warranted and might aid in the development of novel
therapeutic modalities for patients with this condition.

Pyroptosis
Pyroptosis is a novel form of programmed cell death that
differs from other types of cell death in that it is dependent on caspase-1 activation.110 Hepatic caspase-1 activation occurs in both bone-marrow-derived Kupffer cells
(macrophages) and hepatocytes during the development
of NASH and alcoholic steatohepatitis; this activation
seems to be mediated through the NLRP3 inflammasome.111,112 Furthermore, treatment with Il1ra has been
shown to ameliorate alcoholic steatohepatitis in mice
and mice lacking components of the inflammasome,
especially the Nlrp3 inflammasome, are protected or
develop less severe liver disease when stressed with established models of dietary-induced NASH.112 In contrast
to the studies that have used various knockout mice, we
developed a conditional mutant Nlrp3 knock-in mouse
strain.113 Notably, pyroptotic cell death was observed
in hepatocytes with a constitutively activated NLRP3
inflammasome.114 Moreover, isolated hepatocytes from
mice with a globally activated Nlrp3-inflammasome
showed a marked increase in the number of cells with
active caspase-1 and cell membrane pores.114 Treatment
with IL1Ra only partially attenuated liver damage. In
addition, increased collagen deposition and activation of
HSCs was found in Nlrp3 knock-in mice, supporting a
finding that HSCs express inflammasome components
and that these components are important in the regulation of HSCs.115 Future studies to dissect the importance
of cell-specific activation of the NLRP3 inflammasome,
and the role of IL1-independent NLRP3 pathways in
liver inflammation, cell death and fibrosis are needed.
Necrosis or oncosis
Unlike the programmed cell death pathways described
above, necrosis (or oncosis) is thought to represent an
accidental form of cell death with simultaneous disruption
634 | NOVEMBER 2013 | VOLUME 10

of multiple pathways.116 Necrosis occurs under many different circumstances in the development of liver diseases:
ATP depletion as a consequence of ischaemia or hypoxic
cell injury; excessive formation of ROS as it occurs
during reperfusion or ischaemic injury; drug-induced
or toxin-induced liver disease by paracetamol or other
xenobiotics; and overwhelming tissue injury as occurs
in acute fulminant liver failure. Often the mode of cell
death by toxic stimuli depends on the concentration, with
lowconcentrations more likely to induce apoptosis and
high concentrations leading to necrosis. Thus, the stimuli
listed above might induce both modes of cell death at
different times, depending on the severity of the insult.116

Conclusions
As the prevalence of NAFLD and NASH continues to rise,
it threatens to become a global epidemic. Over the past
decade, there has been a tremendous growth in research
focus on the molecular mechanisms involved in development of hepatic steatosis and progression to NASH and
fibrosis. These efforts have translated into novel insights
into the disease process that have uncovered a number of
potential targets for designing new therapies and tailoring these interventions for personalized medicine. The
evidence for crosstalk between certain dietary components, gut microbiota and host genetics in the context of
weight gain and obesity has revealed the need to design
integrative approaches to better understand the influence
of these factors on the liver. Moreover, studies have shed
light on the role of innate immune activation and inflammation in progression to more severe forms of NAFLD.
Finally, studies have demonstrated that the way the liver
handles excess lipids and the variety of different lipid
types and metabolites that can accumulate during this
process are important determinants of how liver homeostasis is affected. A number of cell death pathways are
activated during NASH development including apoptosis,
autophagic cell death, necrosis, necroptosis and pyroptosis, which might interact or have redundant roles in
triggering liver damage and fibrosis. Determining these
pathways and their relative importance has the potential
to lead to a new era in our understanding of the pathogenic mechanisms responsible for NAFLD progression
and might promote the development of novel diagnostic
markers, as well as more rational treatment strategies to
halt NAFLD progression.
Review criteria
A search for original articles and reviews published
between 1998 and 2013 and focusing on NAFLD was
performed in PubMed. The search terms used were
NAFLD, NASH, polyunsaturated fatty acids,
fructose, genetics, gut microbiota, innate
immunitiy, toll-like receptors (TLR), inflammasome,
natural killer T (NKT) cells, cell death, apoptosis,
autophagy, necroptosis, pyroptosis, oncosis,
necrosis, IL1, and caspase1, alone and in
combination. All articles identified were English-language,
full-text papers. We also searched the reference lists of
identified articles for further relevant papers.

www.nature.com/nrgastro
2013 Macmillan Publishers Limited. All rights reserved

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Acknowledgements
Work by the authors of this Review was funded by NIH
(DK076852 and DK082451 to A.E. Feldstein, AI52430
to H.M. Hoffman and T32AI007469 to L.Broderick)
and German Research Foundation (DFG-grant 173/21
to A. Wree) grants.
Author contributions
The authors contributed equally to all aspects
ofthisarticle.
Supplementary information is linked to the online
version of the paper at www.nature.com/nrgastro.

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