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Liver Int. Author manuscript; available in PMC 2016 April 08.
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Liver Int. 2016 April ; 36(4): 480487. doi:10.1111/liv.13009.

The enteric microbiome in hepatobiliary health and disease

James H. Tabibian1, Cyril Varghese2, Nicholas F. LaRusso3, and Steven P. O'Hara3
1Division of Gastroenterology, University of Pennsylvania, Philadelphia, PA, USA
2Department of Internal Medicine, Mayo Clinic, Rochester, MN, USA
3Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, USA
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Abstract
Increasing evidence points to the contribution of the intestinal microbiome as a potentially key
determinant in the initiation and/or progression of hepatobiliary disease. While current
understanding of this dynamic is incomplete, exciting insights are continually being made and
more are expected given the developments in molecular and high-throughput omics techniques. In
this brief review, we provide a practical and updated synopsis of the interaction of the intestinal
microbiome with the liver and its downstream impact on the initiation, progression and
complications of hepatobiliary disease.

Keywords
cholangiopathies; cirrhosis; liver diseases; microbiota; toll-like receptors
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Harbouring roughly 2-5 1011 bacteria per gram of faeces in humans, the intestinal tract
embodies an incredibly complex biological ecosystem (1). This microbiome' includes not
only bacteria, which may be symbiotic or pathologic but also the collective ensemble of
fungi, viruses and other microorganisms, their metabolites, degradative byproducts and
genomes, as suggested by the Greek root words bios (life) and oma (mass). The composition
of the human enteric microbiome is influenced by numerous factors including, but not
limited to, host genotype, diet, age, disease state and exposure to antibiotics. The
constituents of the microbiome, in addition to executing their respective biologically
constitutive processes, are known to interact with each other as well as with their host; these
interactions influence innumerable physiologic as well as pathophysiological immuno-
biological processes in a multitude of cell types and tissues (2).
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Receiving approximately 75% of its blood supply from the splanchnic circulation, the liver
is continuously exposed to a wide repertoire of molecules, be they beneficial or noxious,
from the intestinal microbiome. An ever-increasing body of literature has begun to shed light
on the role of this gut-liver axis' in the maintenance of health as well as in the pathogenesis

Correspondence; Dr Steven P. O'Hara, Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, 200 First
Street, SW, Rochester, MN 55905, USA, Tel: +1 507 284 1006; Fax: +1 507 284 0762 ohara.steven@mayo.edu.
James H. Tabibian and Cyril Varghese are co-first authors.
Conflicts of interest: The authors do not have any disclosures to report.
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of fatty liver disease, autoimmune and idiopathic liver disease, hepatic fibrosis and
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hepatocarcinogenesis.

Herein we provide an overview, from both a clinical and molecular perspective, of the
interaction of the intestinal microbiome with host immunity and hepatic physiology as well
as its impact on the initiation, progression and clinical complications of hepatobiliary
disease.

The intestinal microbiome and the healthy liver

The liver maintains robust innate immune capacity which functions as a crucial surveillance
and elimination system in normal physiologic conditions. While certain microbially derived
molecules and other antigens (e.g. metabolites of ingesta) are tolerated, the cells of the liver
simultaneously operationalize immune responses against potentially injurious stimuli (3, 4).
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Vital to this function are pattern-recognition receptors (PRRs), which are plasma membrane
and cytoplasmic proteins that sense and respond to pathogen-associated molecular patterns
(PAMPs). PRRs, which include toll-like receptors (TLRs) and nucleotide-binding
oligomerization domain receptors (NLRs), have been identified in numerous recruited and
resident hepatic cells including biliary epithelial, sinusoidal, endothelial, stellate, Kupffer
cells and hepatocytes (58).

Thirteen TLRs have been identified to date in mammals, 10 of which have been
characterized in humans (i.e. TLR1-10). These type 1 transmembrane proteins are located on
the plasma membrane or in the endosomal compartment (i.e. TLR3, TLR7 and TLR9) (9)
each recognizing distinct lipid, lipoprotein, protein and nucleic acid molecular patterns from
microbes such as bacteria, virus, parasites and fungi. TLRs initiate signalling cascades
through at least two pathways, MyD88-dependent and MyD88-independent pathways.
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Recognition of individual PAMPs induces distinct signalling pathways via hetero- and
homodimerization of TLRs, the recruitment of distinct adaptor proteins and activation of a
multitude of downstream signalling molecules (Fig. 1). TLR signalling in the liver is
activated when PAMPs are delivered to it, for example, via the portal venous circulation,
hepatic artery or ascension from the small intestine, and recognized by their respective
cognate TLRs (8). This leads to signal transduction culminating in nuclear translocation of
transcription factors and ultimately biosynthesis of interleukins, interferons and/or
chemokines, including but not limited to tumour necrosis factor (TNF-), interleukin (IL-1)
and various interferons (Fig. 1). Persistent TLR activation facilitates the clearance of PAMPs
but may also lead to secondary hepatobiliary injury (e.g. inflammation, fibrosis and cellular
senescence) depending on the nature of the insult/PAMP as well as host immunogenetic
factors (3, 10).
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Nucleotide-binding oligomerization domain receptors are cytoplasmic proteins which can

work in conjunction with TLRs. To date, 22 NLRs have been identified in humans. Similar
to TLRs, recognition of ligand (i.e. a PAMP) by an NLR results in a cascade of signalling
events leading to specific cytokine production as well as other resident and recruited cellular
responses. A specific subfamily of NLRs is able to assemble into an inflammasome, that is,
a multiprotein complex comprised of a sensor protein, adaptor protein and effector enzyme

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(caspase). Inflammasomes recognize PAMPs as well as endogenous damage-associated

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molecules (e.g. ATP), and upon stimulus, can trigger autocatalytic cleavage of caspase-1
with subsequent bioactivation of interleukin 1p and IL18 (Fig. 1) (3, 11).

Additional innate immune PRRs present in the liver include the retinoic acid-inducible gene
1 (RIG-I)-like receptors (RLRs) and the C-type lectin receptor (CLR) family (6, 12). RLRs
(e.g. RIG-I, MDA5 and LGP2) are cytoplasmic proteins involved in viral recognition and the
induction of a type-1 interferon response, while CLRs comprise a large family of calcium-
dependent PRRs involved in fungal recognition and initiation of multiple innate immune
responses (12, 13).

Given the intricate nature of PRR signal transduction and its downstream effects, it is not
surprising that defects in these processes are associated with a variety of infectious,
autoimmune and metabolic disorders, including those of the hepatobiliary system (14).
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In addition to microbial molecules, it is also worth mentioning microbial metabolites (and

their receptors) in the context of hepatobiliary health and disease. Such metabolites include
but are not limited to: (i) bile acid (BA) homoeostasis and the farnesoid X receptor [FXR;
the activation of which leads to transcription of fibroblast growth factor 19 (FGF19), a
regulator of BA synthesis] (1517), (ii) short-chain fatty acids and their respective receptors
(18, 19) and (iii) phosphatidyl choline from dietary choline (15, 20, 21). Understanding of
the roles of these mediators of cellular signalling is an area of active biomedical and
potentially therapeutic investigation.

The intestinal microbiome as a potential etiologic trigger in hepatobiliary

disease
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Multiple lines of evidence suggest that the microbiome may play a primary role in the
initiation of select liver diseases. Multiple mechanisms have been proposed regarding how
microbial and other intestinally derived molecules may incite hepatobiliary injury which
may lead to chronic disease. Below we mention a few examples of hepatobiliary disorders
wherein the intestinal microbiome may be a key etiologic trigger.

Primary sclerosing cholangitis

Perhaps the best evidence for an enteric microbial aetiology of hepatobiliary disease is
embodied in primary sclerosing cholangitis (PSC), a chronic, cholestatic, fibroinflammatory
cholangiopathy (3, 10, 22). It is well known that nearly 75% of patients with PSC also have
inflammatory bowel disease (primarily ulcerative colitis), a disorder which itself is
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associated with intestinal dysbiosis (23). In addition, portal bacteraemia, bactero- bilia (24,
25) and 16s ribosomal ribonucleic acid (rRNA) in bile (26, 27) have all been described in
PSC. Moreover, PSC cholangiocytes accumulate lipopolysaccharide (LPS) in vivo (28) and
are hyper-responsive to LPS treatment in vitro (22, 29, 30). In addition, several genomic
associations have been established with loci implicated in host-microbiome interactions (e.g.
fuco-syltransferase 2, interleukin 2 receptor) in patients with PSC (3133). Intriguingly,
recent studies have shown that treatment with select oral antibiotics may offer several
therapeutic benefits in patients with PSC (10, 34, 35) Further evidence for an aetiologic role

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of the intestinal microbiome is provided by animal model studies, wherein enteric microbial
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molecules/dysbiosis have been shown to lead to hepatobiliary injury with several

characteristic features of PSC (10).

Primary biliary cirrhosis

There is some evidence for a primary role of the micro- biome in other liver diseases as well.
For example, in primary biliary cirrhosis (PBC), antimitochondrial antibodies (seen in
95% of patients with PBC) exhibit cross reactivity to bacterial antigens from Escherichia
coli and Novosphingobium aromaticivorans, and PBC cholangiocytes can accumulate LPS
in vivo (28). Furthermore, susceptible strain mice inoculated with N. aromaticivorans have
been shown to develop PBC-like disease. Collectively, these and other findings indicate that,
as with PSC, a gut-liver axis disruption may serve as a potential aetiologic trigger in the
pathogenesis of PBC (36, 37) It is unclear, however, if these microbiome-related findings
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represent the cause, result or only associated markers of hepatobiliary disease.

The intestinal microbiome as a modulator of chronic hepatobiliary disease

There is a growing body of research which suggests that the intestinal microbiome can
impact the features and progression of existing hepatobiliary disease. Ostensibly the best
data in this regard have thus far have come from studies of non-alcoholic fatty liver disease
(NAFLD) (38), but an increasing number of studies have implicated the intestinal
microbiome in other hepatobiliary disease processes as well.

Non-alcoholic fatty liver disease

It is estimated that NAFLD is prevalent in 30% of the general population and 75% of obese
individuals in the USA. Nearly a quarter of these patients will develop non-alcoholic
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steatohepatitis (NASH), of which approximately a third will progress to cirrhosis (39, 40). It
is, therefore, not an overstatement that NAFLD (and in particularly NASH) represents a
major public health concern. Among the many aspects of NAFLD/NASH that have been
investigated to date, there are accumulating data that the intestinal microbiome is altered in
patients with this disease. For example, the abundance of the two major intestinal bacterial
phyla, Firmicutes and Bacteroidetes, is altered, with most studies revealing a relative
increase in the former (41). The altered micro- biome in NAFLD has an increased capacity
to harness energy from the host diet and can suppress fasting- induced adipocyte factor (i.e.
ANGPTL4), thereby increasing lipoprotein lipase activity and hepatocyte triglyceride
accumulation (42). In addition, high-fat diets lead to microbiome changes that increase
conversion of dietary choline into methylamines. Hence, plasma concentrations of
phosphatidylcholine are reduced, which in turn decreases the assembly and secretion of very
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low density lipoprotein (thereby promoting triglyceride accumulation in hepatocytes) (21).

There are also changes in the metabolites of the altered intestinal microbiome in NAFLD
(21); for example, ethanol production by intestinal bacteria is increased, which further
promotes hepatocyte triglyceride accumulation and induces a second hit' to the liver via
generation of reactive oxygen species (43). Another example is that of deoxycholic acid, a
bacterial metabolite of cholic acid, which has recently been linked to obesity-associated
hepatocellular carcinoma (44). It is worth mentioning here that BAs, in addition to being

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acted upon enzymatically by intestinal bacteria, can also have direct (antimicrobial) effects
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on the bacteria themselves, both directly as well as indirectly through FXR-mediated

antimicrobial peptides (16). In animal models, high-fat, high-fructose, methionine/choline-
deficient diets cause an increase in serum LPS levels, indicating that diet influences the
intestinal microbiome and (consequently) permeability (45, 46). Corroborating these data is
the finding of alterations in enterocyte tight junctions and increased intestinal permeability
in patients with NAFLD (47), which may serve as a means for greater enterohepatic
circulation of potentially injurious intestinal molecules. Additionally, mice fed a high-fat
diet exhibit hyper-responsivity to low-dose LPS administration, including increased pro-
inflammatory cytokine expression, elevated liver triglyceride content and inflammatory cell
infiltration (48). In this murine model, obesity-induced leptin concentrations appear to
promote, via STAT3, increased Kupffer cell expression of CD14 (a receptor for LPS), thus
sensitizing these cells to endotoxin. Together, these intriguing results suggest that not only is
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NAFLD associated with increased circulating endotoxin levels but may be a condition in
which hepatic innate immune cells are (inordinately) primed for responding to circulating
endotoxin.

Alcoholic liver disease

A growing number of studies have found an association between the intestinal microbiome
and alcoholic liver disease. Alcohol is known to cause intestinal bacterial overgrowth in
humans as well as mouse models. Indeed, jejunal aspirates from patients with chronic abuse
appear to harbour greater numbers of bacteria compared to controls (49). Chronic alcohol
ingestion is also associated with qualitative disturbances in the intestinal microbiome, such
as decreased commensal bacteria (e.g. Lactobacilli), among other dysbiotic alterations (50).
Alcohol, being a chemical solvent, also increases intestinal permeability (51, 52) as do its
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metabolites, which may further predispose to liver injury as a result of increased

enterohepatic circulation of dysbiotic microbial molecules (as in NAFLD). In experimental
alcoholic liver disease, mice deficient in TLR4 or treated with antibiotics have been shown
to exhibit decreased alanine aminotransferase levels, steatosis and inflammation compared to
wild-type alcohol fed mice (53). These experimental results are consistent with the hepatic
innate immune response to endotoxin via TLR4 activation in early alcohol-induced liver
injury. More recently, it has been shown that alcohol administration directly reduces the
capacity of the murine microbiome to synthesize nearly all saturated long-chain fatty acids
(LCFAs) (54). Moreover, this depletion of saturated LCFAs correlates with a decrease in
Lactobacilli, which utilize LCFAs in vivo for growth. Importantly, dietary administration of
saturated LCFA, but not unsaturated LCFA, appears to restore barrier function and prevent
alcohol-induced liver injury. Hence, and somewhat in contradiction to what is observed in
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NAFLD, saturated LCFAs, both dietary and microbially generated, exhibit a protective effect
in alcoholic liver disease, likely through restoration of eubiosis and restoration of the
intestinal epithelial barrier.

Other hepatobiliary diseases and processes

There is preliminary evidence that the intestinal microbiome may influence the pathogenesis
of other, and perhaps somewhat unexpected diseases, including but not limited to chronic
viral hepatitis (20) and autoimmune hepatitis (55). The microbiome may also contribute to

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(or delay) progression of pathobiological hepatic processes such as inflammation, fibrosis

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and neoplasia (17, 44, 56). For example, deficiency in TLR4 signalling and antibiotic
treatment have both been shown to reduce hepatic fibrosis following experimental bile duct
ligation (57). With the above in mind, the impact of the microbiota in the modulation of
chronic liver disease may well be far-reaching and is a topic of ongoing and important
investigation (52).

The intestinal microbiome and its role in complications of advanced

hepatobiliary disease
The common histologic sequela of chronic hepatic inflammation because of any underlying
disease process is hepatic fibrosis and ultimately cirrhosis. Intestinal microbial dysbiosis in
cirrhosis is facilitated by several pathophysiologic processes including decreased BA
production, defects in innate immunity, impaired intestinal motility and increased intestinal
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congestion as a result of reduced portal venous flow. Prior studies have shown significant
over-representation of Streptococcaceae and underrepresentation of Lachnospiraceae in
patients with cirrhosis (58), although specific changes may depend on various host factors as
well as the type and severity of underlying disease (59).

Complications of cirrhosis are in some ways related to the intestinal microbiome. Increased
ammonia causing hepatic encephalopathy is because of both liver dysfunction and possible
overproduction of ammonia by a dysbiotic microbiome. Thus, it is no surprise that intestinal
decontamination with non-absorbable antibiotics, such as rifaximin, is an effective treatment
for subclinical hepatic encephalopathy (60). Spontaneous bacterial peritonitis, another
complication of cirrhosis, occurs as a result of migration of intestinal bacteria into the
peritoneal cavity, the risk of which increases with advancing cirrhosis because of a
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combination of effects including increased intestinal permeability and bacterial overgrowth.

As with hepatic encephalopathy, the standard treatment for spontaneous bacterial peritonitis
includes antibacterial therapy.

The hepatoprotective role of the commensal microbiome

While intestinal microbial dysbiosis and certain pathologic microbial molecules may initiate
or exacerbate hepatobiliary injury, a growing and exciting body of literature suggests that the
commensal microbiota may have protective effects against hepatobiliary injury and disease.
For example, a recent study demonstrated more severe injury, including but not limited to
hepatic fibrosis, in germ-free (GF) mice treated with carbon tetrachloride or thioacetamide
(61). Intriguingly, in this same study, a similarly severe phenotype was observed in MyD88/
TRIF-deficient mice exposed to these hepatotoxins, suggesting that the lack of innate
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immune signalling was a key determinant of increased liver injury. In an even more recent
study, we observed significantly exacerbated serological and histological hepatobiliary
disease as well as increased cholangiocyte senescence in GF compared to conventionally
housed mdr2/ mice (62). Furthermore, we found that the commensal microbial metabolite
ursodeoxycholic acid (which was absent in GF mice) abrogates human cholangiocyte
senescence in an in vitro model of LPS- or H202-induced cellular senescence (62).

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It is now recognized that both primary (hepatocyte- derived) and secondary (microbially
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modified) BAs function as signalling molecules. BAs regulate their own synthesis and
modulate key metabolic pathways and inflammation through activation of FXR
(preferentially through primary BAs) and the G protein-coupled BA receptor 1 [GPBAR1
(TGR5); preferentially through secondary BAs] (63, 64). While the composition of the BA
pool is modulated by microbial metabolism of primary (hepatocyte-derived) to secondary
BAs, the composition of the microbiome is modulated by BAs (65, 66). This delicate
balance of BA composition, microbial populations and the activation of FXR and TGR5 has
profound effects on metabolic, anti-inflammatory and hepatoprotective processes in the liver.
For this reason, FXR and TGR5 agonists continue to be explored as potential therapeutic
approaches for liver disease in both animal models (reviewed in 67) and in clinical trials
(reviewed in 68). The aforementioned and other accumulating findings provide proof of
concept regarding the protective role of the commensal microbiota and its metabolites in the
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pathobiology of hepatobiliary injury and disease. Future studies should aim to better
understand the structure and role of the commensal microbiota in hepatobiliary disease and
identify commensal microbial metabolites which may impart cyto- protective effects against
hepatobiliary injury. Insights from such studies are expected to generate new knowledge
regarding how to harness the therapeutic properties of the commensal microbiota for
translation into potential therapeutic applications.

Conclusion
The large biomass of microorganisms and molecules comprising the human intestinal
microbiome has constant interactions with the liver (conceptual schema presented in Fig. 2).
Collectively, these organisms have significant effects on the development, progression and
outcomes of hepatobiliary disease, the extent and nature of which remain areas of ongoing
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investigation and intrigue. In this brief review, we have provided a framework which
delineates current understanding of the role of the microbiome in health as well as various
stages of hepatobiliary disease. Although these relationships and multidirectional
interactions can be incredibly complex, with the advent of high-throughput techniques and
increasing reliance on a team science approach, the ability to rapidly, reliably and affordably
study these dynamics will continue to grow. These advances hold promise in elucidating
pathophysiological ramifications in hepatobiliary health and disease as well as identifying
novel therapeutic opportunities.

Acknowledgments
Financial support: This work was supported by National Institutes of Health Grants AI089713 (to S.P.O.),
DK57993 (to N.F.L), the Mayo Foundation, and the Mayo Clinic Center for Cell Signaling in Gastroenterology
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(P30DK084567).

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Abbreviations

BA bile acid
CLR C-type lectin receptor
FGF19 fibroblast growth factor 19
FXR farnesoid X receptor
GF germ free
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GPBAR1(TGR5) G proteincoupled bile acid receptor 1

LCFA long-chain fatty acids
LPS lipopolysaccharide
NAFLD non-alcoholic fatty liver disease
NASH non-alcoholic steatohepatitis
NLRs nucleotide-binding oligomerization domain receptors
PAMPs pathogen-associated molecular patterns
PBC primary biliary cirrhosis
PRRs pathogen-recognition receptors
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PSC primary sclerosing cholangitis

RLRs retinoic acid-inducible gene receptors
TLRs toll-like receptors
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Key points
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The intestinal microbiome and the gut liver axis' are increasingly recognized in
the maintenance of hepatobiliary health as well as the pathogenesis of disease.

The liver plays an important role in innate immunity, defects in which are
associated with a variety of hepatobiliary disorders.

Perturbations in the intestinal microbiome may lead to the initiation of select

liver diseases and/or impact the features and progression of existing hepatic
disease.

Host-microbiome interactions are incredibly complex, yet with the advent of

high-throughput technologies and team science approaches, our understanding
of these dynamics will continue to grow.
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Fig. 1.
Schematic representation of TLR and NLR pathways driving pro-inflammatory state after
exposure to extracellular and Intracellular microbial products. TLRs are localized either on
the cell surface (TLR 2, 1,4, 6, 11) or within an endosome (TLR 3, 7, 9) of biliary epithelial,
sinusoidal, endothelial, stellate, Kupffer cells and hepatocytes. TLR 2, 1 and 6 associate with
each other and adaptor proteins, thereby initiating (i) MyD88 adaptor protein-dependent
NFB activation and subsequent transcription of inflammatory cytokines (e.g. IL-1 and
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IL-18). TLR3 and TLR4, when stimulated, also initiate (ii) the TRIF pathway, which
activates downstream IRFs and subsequent transcription of type I interferons. NLRs such as
NOD1/2, when stimulated by intracellularly incorporated signals (e.g. bacterial
peptidoglycans), form inflamma- some complexes that lead to both (iii) NFB activation and
caspase production, the latter of which (iv) activates precursor cytokines, e.g. IL- 1 and
IL-18. IL, interleukin; IRF, interferon regulatory factor; NLR, NOD-like receptor; NOD,
nucleotide-binding oligomerization domain; TLR, toll-like receptor; TRIF, TIR-domain-
containing adapter-inducing interferon-.
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Fig. 2.
Conceptual schema of the gut microbiome-liver interaction in hepatobiliary disease. A
healthy, highly diverse gut microbiome maintains liver health through the synthesis of
metabolites involved in immune regulation, lipid and BA homoeostasis, and energy
utilization (cytoprotective). The physiology and dysregulation of this gut-liver axis and its
complex and dynamic interactions are the focus of intense investigation and hold promise
for advancing management of chronic liver disease. Additionally, a growing body of basic,
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translational, and clinical evidence suggests that intestinal microbiome dysbiosis and
microbially derived molecules (1) modify the microbial environment and may be involved in
the progression of chronic hepatobiliary disease. Multiple mechanisms may be involved
including the disease, host genotype, diet, age and exposure to antibiotics. In disease models
and patients with advanced cirrhosis, there is (2) epithelial barrier defects (e.g. tight junction
disruption). This results in increased intestinal permeability and enterohepatic circulation of
injurious microbial molecules (cytodestructive, e.g. lipopolysaccharide). In the liver, these
PAMPs (3) activate PRRs on multiple cell types including hepatocytes, biliary epithelia,
endothelial, stellate and Kupffer cells. Activation of PRRs (4) induces the expression of pro-
inflammatory mediators and promotes hepatobiliary injury (e.g. inflammation, fibrosis,
apoptosis and senescence) and initiation of immune dysregulation (e.g. development of auto-
immunity). Ultimately, these processes may result in the (5) induction, modulation or
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progression of chronic liver disease. BA, bile acid; NLR, NOD-like receptor; PAMPs,
pathogen-associated molecular patterns; PtdC, phosphatidylcholine; TJ, tight junction; TLR,
toll-like receptor. Figure modified from ref. (69).

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