Download Novel therapeutic targets in primary biliary cirrhosis

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Novel therapeutic targets in primary
biliary cirrhosis
Jessica K. Dyson, Gideon M. Hirschfield, David H. Adams, Ulrich Beuers, Derek A. Mann,
Keith D. Lindor and David E. J. Jones
Abstract | Primary biliary cirrhosis (PBC) is a chronic immune-mediated liver disease characterized by
progressive cholestasis, biliary fibrosis and eventually cirrhosis. It results in characteristic symptoms with
marked effects on life quality. The advent of large patient cohorts has challenged the view of PBC as a benign
condition treated effectively by the single licensed therapy—ursodeoxycholic acid (UDCA). UDCA nonresponse
or under-response has a major bearing on outcome, substantially increasing the likelihood that liver
transplantation will be required or that patients will die of the disease. In patients with high-risk, treatmentunresponsive or highly symptomatic disease the need for new treatment approaches is clear. Evolution in our
understanding of disease mechanisms is rapidly leading to the advent of new and re-purposed therapeutic
agents targeting key processes. Notable opportunities are offered by targeting what could be considered as
the ‘upstream’ immune response, ‘midstream’ biliary injury and ‘downstream’ fibrotic processes. Combination
therapy targeting several pathways or the development of novel agents addressing multiple components of
the disease pathway might be required. Ultimately, PBC therapeutics will require a stratified approach to be
adopted in practice. This Review provides a current perspective on potential approaches to PBC treatment,
and highlights the challenges faced in evaluating and implementing those treatments.
Dyson, J. K. et al. Nat. Rev. Gastroenterol. Hepatol. 12, 147–158 (2015); published online 3 February 2015; doi:10.1038/nrgastro.2015.12
Introduction
Institute of Cellular
Medicine, 3rd Floor
William Leech Building,
Medical School,
Framlington Place,
Newcastle University,
Newcastle upon Tyne
NE2 4HH, UK (J.K.D.,
D.A.M., D.E.J.J.).
Centre for Liver
Research, NIHR
Biomedical Research
Unit, University of
Birmingham, Wolfson
Drive, Birmingham
B15 2TT, UK (G.M.H.,
D.H.A.). Department
of Gastroenterology
& Hepatology, Tytgat
Institute for Liver and
Intestinal Research,
Academic Medical
Center, G4‑216,
University of
Amsterdam,
PO Box 22600,
NL‑1100 DD,
Amsterdam,
Netherlands (U.B.).
College of Health
Solutions, Arizona
State University,
550 North 3rd Street,
Phoenix, AZ 85004,
USA (K.D.L.).
Correspondence to:
J.K.D.
jessicadyson@
doctors.org.uk
Primary biliary cirrhosis (PBC) is a chronic immunemediated liver disease characterized by damage to,
and destruction of, the biliary epithelial cells (BECs) and
small intrahepatic bile ducts, leading to progressive
cholestasis, biliary fibrosis and eventually cirrhosis.1
PBC, which has a prevalence of up to 35 per 100,000 in
Europe,2 affects patients through the development of cirrhosis with its attendant complications and risk of death,
and through the development of characteristic symptoms
including pruritus and fatigue with substantial effects on
quality of life.3,4
PBC was, until the past 5 years or so, perceived to
be a typically benign condition of those >50 years old,
effectively treated by a single cheap and safe licensed
therapy, ursodeoxycholic acid (UDCA). The advent of
Competing interests
G.M.H. is an investigator and/or consultant for Biotie Therapies,
Bristol–Myers Squibb, Falk Pharma, FF Pharma, Gilead,
GlaxoSmithKline, Intercept, Johnson & Johnson and NGM
Biopharmaceuticals; he is a co-investigator for UK‑PBC, supported
by a stratified medicine award from the UK Medical Research
Council. U.B. has consultant agreements via the University of
Amsterdam with Intercept and Novartis, and receives lectures
fees from the Falk Foundation. D.A.M. is a consultant and
collaborator with GlaxoSmithKline, Medimmune, Novartis and
UCB. K.D.L. is an unpaid member of the advisory board for
Intercept and Lumena and a paid consultant for Abbvie and
Gilead. D.E.J.J. receives UK‑PBC research and trial funding from
GlaxoSmithKline, Intercept and Lumena, and acts as a consultant
via Newcastle University for Intercept, Johnson & Johnson and
Novartis. The other authors declare no competing interests.
national and international patient cohorts has, however,
challenged this view,4 identifying substantial unmet clinical need,5 particularly in patients showing inadequate
biochemical response to UDCA (which includes up to
30% of patients, rising to >50% in patients presenting
under the age of 40 years4). Poor biochemical response
to UDCA has a major effect on outcome, substantially
increasing the risk of death or need for liver transplantation.6,7 In addition, UDCA treatment has little effect
on the characteristic symptoms of PBC such as pruritus
and fatigue.3,4 Awareness of unmet need has triggered
marked interest in second-line therapy for PBC. This
Review provides a current perspective on potential new
approaches to treatment in PBC, and highlights some
of the challenges we face in evaluating and effectively
implementing those treatments.
Current status of second-line therapy
At present, no second-line therapies for patients with
inadequate response to UDCA therapy have been
approved. A number of agents have, however, undergone evaluation in patients stratified for nonresponse to
UDCA. With the availability of new drugs on the horizon,
the question of tailoring therapy to individual patients
will become progressively more important.
Three interlinked biological processes are at play in
PBC (Figure 1), with drug development activity related
to each. The initial pathogenic insult is conventionally
thought to be targeted at BECs, and to be immunological
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Key points
■■ Primary biliary cirrhosis (PBC) leads to progressive cholestasis, biliary fibrosis
and cirrhosis and characteristic symptoms with a marked effect on life quality
■■ Ursodeoxycholic acid (UDCA) nonresponse or under-response has a
major effect on outcome, substantially increasing the likelihood that liver
transplantation will be required, or that patients will die of the disease
■■ Patients with high-risk, treatment-unresponsive or highly symptomatic
disease need new treatment approaches; as yet, no agents demonstrating
disease‑modifying actions have been shown to improve systemic symptoms
■■ Excellent opportunities are offered by targeting the immune response, biliary
injury and fibrotic processes
■■ Combination therapies with a stratified approach, in which specific treatments
are targeted at the pathological processes at play, or single agents with
multiple actions, are likely to be needed
in nature. Broadly acting immunosuppressive therapies
have, however, proved disappointing to date (at least when
used in a non-stratified manner) and have not entered
routine use (Supplementary Table 1 online). A number of
these agents and approaches might warrant re-evaluation
under the stratified therapy model that targets secondline therapies at UDCA nonresponders. Furthermore,
interest in targeted immunosuppression has increased
following the characterization of the role of immunoregulation in PBC risk8,9 and improved application of disease
stratification,4,10 and a number of trials of biologic agents
are underway. Approaches have included ustekinumab
(NCT0138997311)—an antibody directed at the common
IL‑12/IL‑23 p40 chain—underpinned­by evidence from
genetic studies and others implicating the IL‑12 and IL‑23
pathways in PBC.12–14 Another agent that has been evaluated is NI‑08091—an anti-CXCL10 monoclonal antibody
(NCT0143042915)—supporte­d by evidence implicating
the CXCR3/CXCL10 chemokine/receptor pair in T‑cell
homing into the liver in PBC.16 A further agent potentially set for PBC studies (NCT0219336017) is FFP104, an
anti-CD40 human mono­clonal antibody; a role for the
CD40/CD40L (CD154) interaction in T‑cell licensing
and B‑cell activation has already been proven, as well as a
p­arallel role for this interaction in BEC apoptosis.18
In addition to T‑cell-directed immunotherapy, B‑celltargeted therapy has undergone evaluation in the form
of the anti-CD20 monoclonal antibody, rituximab
(NCT00364819 19). Benefit, albeit very limited, has
been shown in terms of improving liver biochemistry
in patients who do not respond to UDCA.20,21 However,
although the safety profile of rituximab in PBC trials
seems benign,20,21 concern remains because of demonstration of disease deterioration in mouse models of PBC
following anti-CD20 therapy,22 and descriptions of colitis
in human trials in other disease settings.23 All biologic
immunotherapy trials to date, however, have evaluated efficacy in patients who did not respond to UDCA
therapy and who could thus be regarded as being in the
second, cholestatic phase of the disease. The difficulty
is that patients present with high alkaline phosphatase
levels, implying that the phase of disease might not be
well distinguished by biochemical markers. A critical question is the extent to which ongoing immune
injury is present in such patients and, if so, whether it
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has reached a level characterized by persistent epithelial
and stromal cell activation that makes it more resistant to
standard immunosuppression. This issue might explain
the disappointing results seen with biologic agents to
date. Alternatively, perhaps trials are not continued long
enough to see an effect owing to the selected end point
being alkaline phosphatase levels. As yet, no ‘diseasemodifying therapy’ (modification of the immune process
in patients with the earliest evidence of immune injury
and prior to UDCA use) has been attempted. Work is
currently ongoing to try to identify biomarkers associated with immune injury risk in PBC to better target
immunotherapy at potentially responsive patients.
After early (or initiating) immune injury, the disease
process seems to combine cholestasis and fibrosis. The
relative role of each of these mechanisms at a given time
in the course of the disease is unknown. The fact that
UDCA seems to be effective and have a major anti­
cholestatic effect suggests that cholestasis has a central
role in disease progression. Current second-line therapy
evaluation in the areas of cholestasis and fibrosis is largely
around two therapy types, farnesoid X receptor (FXR)
agonists (in particular obeticholic acid, which has shown
efficacy in both phase II and III evaluation)24,25 and peroxisome proliferator-activated receptor‑α (PPARα) agonists
(bezafibrate and fenofibrate), which have shown efficacy
in case reports and series, and which are currently undergoing phase III evaluation (NCT0165473126).27,28 The
fibroblast growth factor‑19 (FGF‑19) analogue NGM282
is also undergoing evaluation (NCT0202640129) and is
relevant given the evidence suggesting that FXR agonists
act in part through FGF‑19 upregulation.30 Other ongoing
trials in PBC are summarized in Table 1.
Future directions in prognostic therapy
Targeting the immune response
If we are to move beyond current therapy, and those agents
currently under evaluation, what are the opportunities for
new target development, and how do they fit into our
understanding of the stages of disease pathogenesis? PBC
is undoubtedly a disease with an autoimmune component;
abundant human and animal data have demonstrated the
central importance of a highly specific and orchestrated
immune response to BECs (Figure 2). Reactivity occurs
at both the T‑cell and B‑cell level to highly conserved
mitochondrial antigens, in particular the E2 component
of the pyruvate dehydrogenase complex (PDC).1,31–33 With
evermore sophisticated approaches to manipulating the
immune system (such as antibody-mediated therapy, targeted inhibitors of cellular pathways relevant to immune
signalling, as well as broader cell therapy approaches aimed
at re-setting an imbalanced immunoregulatory axis) a
renewed opportunity exists for future PBC therapy to be
immune-focused (Figure 3). Such efforts are underpinned
by an increasingly robust set of insights into the initial
immune insults seen in patients with PBC, fundamentally
revolving around a failure of immunoregulation. The PBC
liver is heavily infiltrated by both CD4+ and CD8+ T cells
that react with PDC‑E2, which can be isolated from biopsy
samples.34,35 A predominant type I cytokine pattern with
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Interventions
Immune injury
Small intrahepatic
bile duct
Immunotherapy
BEC
Accumulation
of bile acids
Impaired
bile flow
Cholestasis
Bile acid therapy
Bile duct loss
Epithelial-to-mesenchymal transition
?
Ductopenia
Antifibrotic therapy
Fibrosis
Cirrhosis
Transplantation
End-stage disease
Figure 1 | Pathway for the current understanding
of |PBC
pathogenesis and
Nature Reviews
Gastroenterology
& Hepatology
potential targets for therapeutic intervention. A key upstream disease process is
immune injury to the bile duct cells of the small intrahepatic bile ducts. Although
autoreactive responses in PBC are characteristically directed against highly
conserved self-antigens (in particular the dihydrolipoamide acetyltransferase [E2]
component of pyruvate dehydrogenase complex), the potential for a secondary
inflammatory response following BEC senescence is now emerging. Following initial
immune injury to BECs, progressive bile duct loss is seen, which leads to impaired
bile flow and retention of bile acids. The predominantly hydrophobic bile acid pool
seen in PBC results in enhanced injury to BECs (hydrophobic bile acids are
cytotoxic) leading to a self-sustaining cholestatic cycle of enhanced bile duct injury
and thus progressive cholestasis. Ductopenia occurs as an end-stage of this
process (potentially compounded by BEC replicative senescence and phenotypic
change through a process of BEC epithelial to mesenchymal transition). Progressive
cholestasis leads to fibrosis development and eventually cirrhosis and end-stage
disease. Abbreviations: BEC, biliary epithelial cell; PBC, primary biliary cirrhosis.
high levels of IFN‑γ, IL‑5, IL‑6, IL‑10, IL‑12 and IL‑15 in
the blood and liver of patients with PBC has been demonstrated.14 The portal tracts in patients with PBC are
rich in chemokines secreted by inflamed cholangiocytes,
including CXCL10, CXCL9, CX3CL1 and CCL20, which
are responsible for recruiting T effector cells that bear their
cognate receptors. Type 1 T helper cells (TH1) and cytotoxic T cells express CXCR3 (for CXCL9 and CXCL10)
and CX3CR1, and TH17 cells express CCR6.36 Although
both T‑cell subsets seem to recognize similar sequences
within the same epitope of PDC‑E2 it is believed that CD8+
T cells have a role in the initial degeneration and death
of cholangio­cytes that aberrantly express PDC‑E2.33,35,37
Moreover, an increase in specific cytotoxic T cells in
the liver compared with the peripheral blood has been
reported, which supports the role of these cytotoxic cells in
the evolution of bile duct injury in PBC.33,35 Furthermore,
animal models have highlighted how T cells and B cells
might be relevant to liver injury (reviewed elsewhere38).
On the basis of data from both animal models
and human studies, a disease model has been developed in which a combination of environmental triggers39–42 in genetically predisposed hosts12,13,43,44 leads to
uncontrolled immune activation targeted against the
highly conserved mitochondrial antigens. The critical
steps in the development of an effector cell response,
including antigen presentation, T‑cell differentiation
and proliferation and recruitment, are all under consideration as novel immune targets1 (Figure 3). In particular,
pathway analysis of the genetic risk loci identified suggests
the importance of the IL‑12 pathway and downstream
JAK–STAT signalling. However, the early disappointing results of therapy targeting this pathway 45 suggest an
ongoing need for more-detailed functional post-genomic
studies, including consideration of the potential importance of epigenetic regulation. The specificity and efficacy
of the i­mmunomodulators being investigated must also
be considered.
The challenge in considering targeting of the immune
response in PBC therefore lies not so much in generating a list of targets, or indeed products, as in trying to
quantify the relevance of any particular pathway in diseased liver tissue. The efficacy of interventions must then
be evaluated, assuming that at the time of trial entry a
patient has sufficient ongoing immune-mediated liver
injury to benefit (as opposed to progressive cholestatic
or fibrotic disease). Without good markers of treatment
efficacy that can provide an early and sensitive indicator of nonresponse to therapeutic intervention there is
a risk that novel immunotherapeutic agents will fall foul
of short-term evaluations in classically designed earlyphase trials that use biochemical end points such as alkaline phosphatase levels, which are of limited relevance to
phases of the disease that are pre-cholestatic (as a number
of such agents might already have done). Conceptually,
specific targeting of the autoimmune response in PBC
could occur at several levels. These different levels include
depletion of culprit cell subsets (for example, anti-CD3
or alemtuzumab therapy), the prevention or modulation
of T‑cell priming (such as interfering with CD80 signalling), modulation of the degree or nature of the resulting T‑cell response through the induction of tolerance
(regulatory T cells or mesenchymal stem cell therapy or
antigen-specific immunotherapy) and the prevention of
recruitment of effector cells into the liver. The challenge
for immunotherapy in PBC is the balance between efficacy of the immunomodulatory action on the disease and
immune-compromise.
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Table 1 | Examples of active clinical trials in PBC
Therapeutic target
Agents
Background
Budesonide
Phase III trial to compare the efficacy and tolerability
of combination therapy with UDCA (12–16 mg/kg
body weight daily) plus budesonide (9 mg daily) vs
UDCA (12–16 mg/kg body weight daily) plus placebo
Depending on ALT values 6 mg daily budesonide
are allowed
Study population is patients with PBC at risk of
disease progression (NCT00746486150)
Budesonide is a corticosteroid agent with high
first-pass liver metabolism, allowing liver action with
limited systemic adverse effects
Biologic agents
Phase IV trial of abatacept to determine if it is
effective in patients with PBC who do not respond
adequately to UDCA (NCT02078882151)
Abatacept consists of a fusion protein of the extracellular
domain of CTLA‑4 and human IgG1, which binds to CD80
and CD86 molecules on the APC and prevents the
co-stimulatory signal being delivered to the T cell that is
needed for an immune response152
Highly active
antiretroviral
therapy
Trial of combination therapy with tenofovir–
emtricitabine and Iopinavir–ritonavir in patients
with PBC (NCT01614405153)
Recruitment was limited owing to poor tolerance
of lopinavir–ritonavir
Some evidence suggests that PBC might occur as a
result of a retroviral infection in patients who are more
susceptible to disease than others
Early studies with combination antiviral therapy showed
some improvement in biochemistry and histology results
A NOD.c3c4 mouse model of autoimmune biliary disease
showed response to combination antiretroviral therapy154–157
Stem cells
A phase I study of nonmyeloablative allogeneic
haematopoietic stem cell transplantation in
patients with PBC (NCT00393185158) was
withdrawn prior to enrolment
The study was designed to examine whether modulation
of the immune system by treating patients with high dose
cyclophosphamide, fludarabine and CAMPATH-1H, followed
by return of blood stem cells that have been previously
collected from a patient’s sibling would stop or reverse
the disease
The stem cell infusion was to restore blood production
after treatment with cyclophosphamide, fludarabine and
CAMPATH-1H, and to produce a normal immune system
Phase I and II study to assess the safety and
efficacy of umbilical cord mesenchymal stem cells
in PBC (NCT01662973159)
Umbilical cord mesenchymal stem cells have been used
to treat several autoimmune diseases, such as immune
thrombocytopenia, systemic lupus erythematosus and
therapy-resistant rheumatoid arthritis
Abbreviations: APC, antigen presenting cell; ALT, alanine aminotranferase; PBC, primary biliary cirrhosis.
There is also the issue of when in the disease course such
therapy should be given. Early, disease-modifying therapy
would be the ideal solution, but this approach is not plausible when broadly acting agents are being considered owing
to our current inability to predict which patients are at risk
of clinically significant and rapid progression and in whom
the benefits of such therapy would outweigh the risks. The
challenge of this balance explains why depleting therapies
have largely not been explored in PBC and why results
with plausible agents such as ustekinumab have been disappointing (as they have perhaps been used too late in the
disease process, or they need to be used in conjunction
with therapies that also modulate cholestasis). Depleting
therapies are most likely to be effective in early disease
before it is clear that a patient is at high risk of progression (given that we lack predictive biomarkers at present).
It is difficult to justify the use of expensive immunemodulati­ng therapies that might have unpredictable longterm consequences in patients in whom the level of benefit
is uncertain. Chemokine targeting therapy is challenging
because of the redundancy of the chemokine networks and
the potential difficulty of finding the correct dose to use
(that is, at a level that can account for the degree of inflammatory activity). Tolerogenic therapy is potentially the
most attractive option, particularly if detailed knowledge
of the nature of the autoantigen in PBC could be used to
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develop antigen-specific tolerogenic approaches. However,
it should be noted that early attempts to use this approach
in patients have proved unsuccessful, albeit not harmful.46
Targeting cholestasis
Bile lies at the heart of PBC and its therapy, given the
nature and identity of the targets for cellular injury, and
the fact that the only proven drug therapy to date is itself
a bile acid. A critical concept in disease pathogenesis
is that injury to the bile duct, regardless of its original
trigger, is cyclical in nature with cholestasis resulting
from bile duct injury potentially causing further bile
duct injury through the actions of retained hydro­phobic
bile acids.47–49 This process potentially provides both a
mechanism for chronic bile duct injury and a therapeutic
opportunity through breaking the cycle. Bile formation is
regulated by a complex signalling network of transcriptional and post-transcriptional mechanisms in hepatocytes and cholangiocytes.50,51 Cholestasis might result
from both hepatocellular and cholangiocellular secretory
defects and obstructive bile duct lesions, and evidence
suggests that both are at play in PBC.
To date, the best studied anti-cholestatic agent in
hepato­cellular cholestasis remains UDCA.52–54 UDCA
has potent anticholestatic and antiapoptotic properties and
is enriched from 1–2% to ~40% of total bile acids in the
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REVIEWS
Cytotoxic T cell
Apoptosis
PDC
component/
epitope
2
Latent
TGF-β
1
ROS
Active
TGF-β
NK cell
?
3
Hydrophobic
bile acid
Bicarbonate
transporter (e.g. AE2)
‘Bicarbonate
umbrella’
Anti-PDC
antibody
Senescence
Apoptosis
EMT?
Figure 2 | Mechanisms of BEC injury
in PBC.
(1) Immune-mediated
injury
via the
Nature
Reviews
| Gastroenterology
& Hepatology
actions of cytotoxic T‑cells reactive with PDC derived epitopes. An alternative
potential mechanism is through antibody-dependent cell cytotoxicity with antibody
reactivity against PDC/PDC cross-reactive proteins expressed on the surface of
BECs in PBC and mediated by natural killer and other effector cells. (2) Toxic effects
of hydrophobic bile acids able to induce apoptosis of BECs owing to the loss of the
protective ‘bicarbonate umbrella’. (3) BEC plasticity resulting from the local actions
of TGF‑β converted from the latent to the active form and triggered by oxidative
stress resulting from the actions of ROS and other entities. Pathways include
senescence and EMT. The three mechanisms are not mutually exclusive, indeed
they might be fully interdependent. Antigen or epitope generation for autoreactive
immune responses might occur as a consequence of the BEC changes resulting
from the hostile environment following failure of the bicarbonate umbrella.
Furthermore, oxidative stress causing BEC plasticity might also result from the
actions of hydrophobic bile acids in the hostile bile duct environment. All postulated
mechanisms offer potential targets for novel therapy development. Abbreviations:
BEC, biliary epithelial cell; EMT, epithelial to mesenchymal transition; NK, natural
killer; PBC, primary biliary cirrhosis; PDC, pyruvate dehydrogenase complex; ROS,
reactive oxygen species; TGF-β, transforming growth factor beta.
bile of patients with PBC and healthy volunteers treated
with therapeutic daily doses of 15 mg/kg body weight.55
How does UDCA exert its anticholestatic properties at the
level of the hepatocyte? In contrast to hydrophobic bile
acids of the type seen to predominate in PBC and thought
to contribute substantially to bile duct injury, UDCA does
not markedly affect transport protein expression in vivo
at the transcriptional level to modulate transport capacity.54 The conjugates of UDCA were unravelled in the
1990s as calcium agonists56–58 and activators of protein
kinase C (cPKCα59–61), ERK1, EKR2, p38 MAP kinase62–64
and integrins.65 The concept that these conjugates might
enhance the secretory capacity of hepatocytes by stimulating vesicular exocytosis and, thereby, insertion of key
carriers such as the bile salt export pump (BSEP)63,66 or
the conjugate export pump (MRP2)67 into their target
membranes was independently developed in healthy
liver in the context of bile-acid-induced hepatocyte swelling 68 and in the cholestatic liver.69 Experimental models
showed that taurine conjugates of UDCA exert choleretic effects by apical carrier insertion via a dual MAPKdependent and ­integrin-dependent mechanism in healthy
liver 63,64 and anticholestatic effects by stimulation of
impaired apical carrier insertion via mechanisms dependent on Ca2+-/type II inositol‑1,3,4-triphosphate receptor,
cPKCα and PKA.66,67,70–72 Optimization of bile acid therapy
in PBC by additional stimulation of signalling related to
secretory mechanisms might be an attractive approach.
In cholangiocellular and obstructive cholestasis in PBC
the precise interplay of different mechanisms of the disease
process is not understood. The model of a ‘biliary bicarb­
onate umbrella’ as a physiological protective mechanism
of bile duct epithelia against the toxic effects of milli­molar
concentrations of glycine-conjugated bile acids in the
human biliary tree has been proposed.73 Experimental confirmation of this hypothesis in vitro74 led to the speculation
that defects in the biliary bicarbonate umbrella might have
a critical role in the development of bile duct lesions in PBC
as well as other fibrosing cholangio­pathies.73,74 An emerging body of clinical and experimental evidence supports
the concept that therapeutic stabilization of the bicarb­
onate umbrella of small intrahepatic bile ductules might be
crucial in the treatment of PBC through anti-inflammator­y
and antifibrotic properties. UDCA stimulates biliary
bicarb­onate secretion in patients with PBC as well as in
various experimental settings.54,75 Biliary bicarbonate
secretion is stimulated by current treatment with UDCA
(13–15 mg/kg daily) alone75 or in combination with obeticholic acid,76 the glucocorticoid receptor–pregnane X
receptor (PXR) agonist budesonide (for which there is
emerging evidence for clinical utility in some patients
with early PBC77,78), or the C23-homologue of UDCA,
nor-UDCA.79–81 Nor-UDCA is the strongest bicarb­onate
secretagogue so far identified in humans.79 In addition, the
PPARα agonists might also stabilize the biliary bicarbonate
umbrella of small intrahepatic bile ductules by enhancing
expression of the apical phospholipid floppase ABCB4,82
thereby stimulating biliary phospholipid secretion82
and mixed micelle formation and lowering the levels of
potentially toxic bile acid monomers in bile. These effects
might, at least in part, explain the observed anticholestatic,
anti-inflammator­y and antifibrotic properties of these
c­ompounds in biliary diseases.1,8,51
Regardless of the cholestatic mechanism, a net result of
cholestasis seems to be a cycle of BEC injury and a consequent proliferation of ductular cells. Critically, BEC
phenotype changes can occur over time, with senescence and epithelial to mesenchymal transition seeming
to occur as linked processes contributing to progressive
ductopenia.83–86 Targeting senescence as a mechanism
for BEC and eventual duct loss is an attractive and novel
therapeutic approach. Notably, the process of senescence
is not passive—it is characterized by an inflammatory
‘senescent secretome’ in vitro and in vivo.86,87 This observation adds another layer of complexity to the interplay
between immune and cholestatic processes in PBC and
the sequencing and combining of therapies.
Targeting fibrosis
As PBC evolves it is increasingly characterized by progressive deposition of fibrotic extracellular matrix and,
ultimately, the development of cirrhosis. The fibrotic reaction in the cholangitic lesions of PBC has distinct features
that are important to recognize, such as the loss of bile
ducts, a profound ductular reaction, development of
periportal hepatitis and the formation of predominantly
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IL-23
Inhibiting
T cell
co-stimulation
p40
p19
Anti-IL-12/IL-23
TH17
IL-12
p40
p35
APC
IL-2
TNF
CD80
CD28
T cell
p50
p65
IL-12R
TYK2 JAK2
PKCθ
MHC-II
TCR
P STAT4 P
NKT cell
NK cell
JAK
inhibitors
M0
macrophage
Cytotoxicity
T H0
IFN-γR
IFN-γ
Maturation
PDC-E2
Apoptotic
bodies
TNF
IFN-γ
IL-18
IL-18R
TH1
JAK
inhibitors
JAK2
JAK1
P STAT1
STAT1 P
CD4
CXCR3
CD8
CXCL10
CCR6
Anti-CXCL10
CCL20
TH17
TNF
IL-6
IRAK1
IRAK4
p50
p65
Mθ
macrophage
CD8
BEC
Myd88
IRF5
IRF5 P
Recruitment
TLRs
B
cell
AMA
TH17
Differentiation
TH0
IL-23R
Figure 3 | The immune response in PBC is multifaceted and involves both adaptive and innate components. Potential
Nature Reviews | Gastroenterology & Hepatology
novel therapeutic targets are shown in red. Breakdown in immune tolerance to PDC occurs as a consequence of molecular
mimicry to bacterial antigen or environmentally modified self-antigen, or in response to exposure to self-PDC modified
within BECs, with antigen presentation by activated dendritic cells and APCs and differentiation of the subsequent cellular
immune response. Critical aspects to the subsequent immune response in PBC include: one, the phenotype of the
resulting CD4+ T‑cell response (with evidence to support increased activity of the TH1 and TH17 compartments); two,
the nature and processes responsible for the effector immune response targeting BEC (including the activity of CD8 +
cells and more innate processes including the action of NK and NKT cells and macrophages); and three, the processes
for recruitment of effector T‑cells into the liver including through the actions of chemokines. Potential targets for
immunotherapy include through T‑cell–APC interaction (blockade of CD80 co-stimulation), alteration of differentiation
pathways through targeting of IL‑12–IL‑23, and blockade of chemokine pathways responsible for recruitment of effector
cells into the liver (CXCL10). Abbreviations: AMA, apical membrane antigen; APC, antigen presenting cell; BEC, biliary
epithelial cell; NK, natural killer; NKT, natural killer T cell; PBC, primary biliary cirrhosis; PDC, pyruvate dehydrogenase
complex; TCR, T cell receptor; TH1, type 1 T helper cell; TH17, type 17 T helper cell; TLR, Toll-like receptor.
portal-to-portal fibrotic lesions. The current mechanistic description for the initiation of fibrosis in PBC is that
T‑cell-mediated and cholestasis-driven ductular injury
results in the release of a variety of fibrogenic mediators from damaged BECs.1 These mediators, including
transforming growth factor (TGF)‑β1, connective tissue
growth factor (CTGF), p­latelet-derived growth factor BB
(PDGF-BB) and endothelin‑1, trigger the activation of
neighbouring portal fibroblasts88 to adopt a myofibroblastic phenotype and secrete fibril-forming collagens.
This process contrasts with the early pathological events
for sinusoidal fibrosis that is typically associated with
alcoholic liver disease and NAFLD. In these diseases, the
cellular events responsible for triggering fibrosis involve
activation of liver macrophages, which then stimulate the
transdifferentiation of perisinusoidal hepatic stellate cells
into fibrogenic myofibroblasts.89 Immunohistochemical
analyses have also documented the contribution of
hepatic stellate cells to the fibrotic process at the later
c­irrhotic stages of the disease.90
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The distinct anatomical and immunological components of PBC pathology, coupled with differences in the
phenotypes of portal fibroblasts and hepatic stellate cells
must not be overlooked when considering the applic­
ability of emerging antifibrotic therapeutics for PBC.
A potential problem with the translation of some of the
emerging antifibrotics into the PBC arena is that their
targets have been discovered and validated in preclinical systems that do not necessarily recapitulate molecular
and cellular events that shape the portal-to-portal and
periductular fibrosis of PBC, for example myofibroblasts
derived from hepatic stellate cells in 2D culture models
or rodent models of chronic liver damage.91 Most in vivo
models do not recapitulate the fibrogenic mechanisms
observed in PBC. Bile duct ligation in rodents is widely
used as a model of cholestasis and is characterized by
bile duct damage, a ductular reaction and initiation of
fibrosis between portal tracts.92 However, this model is
an acute model that lacks intrinsic immune-mediated
biliary injury, and the role of portal fibroblasts as a source
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of myofibroblasts in this model is highly contested by
lineage tracing studies.93,94 A second rodent model of
cholestasis is the Mdr2 (Abcb4)–/– mouse, which spontaneously develops histological features of sclerosing cholangitis, including the onion-skin-like periductal fibrosis
also seen in some patients with PBC.95 The spontaneous
fibrogenic mechanism in these animals is triggered by
regurgitation of bile into the portal tracts leading to portal
inflammation and the activation of portal fibroblasts.96
Hence, mechanistically the model more closely resembles
human primary sclerosing cholangitis, but has relevance
to PBC when the aim is to validate therapeutics that target
portal fibroblast-derived myofibroblasts.
Of the emerging antifibrotics currently in clinical
studies, inhibitors of the integrin αVβ6 and lysyl oxidase
homolog 2 (LOXL2) probably have the best potential to
ameliorate disease progression in PBC. αVβ6 is expressed
during epithelial repair, in which it has a pivotal role in
the local activation of TGF‑β1.97 Although the appearance
of αVβ6 expression on hepatic epithelia is not disease
specific it is a feature of surface bile duct epithelia and
transitioning hepatocytes in PBC.98 Encouraging preclinical studies have reported a protective effect of αVβ6
inhibition in the bile duct ligation and Mdr2 (Abcb4)–/–
models.99 A humanized monoclonal antibody, STX‑100,
which targets αVβ6, has advanced to phase II trials in
idiopathic pulmonary fibrosis (NCT01371305100) and
chronic allograft nephropathy (NCT00878761 101).102
LOXL2 is an enzyme that promotes collagen and elastin
cross-linking and in experimental models has been
shown to be important for the progression and maturation of fibrosis.103 LOXL2 expression is enhanced in PBC
as well as in other liver diseases, and there is great interest in the outcome of ongoing trials with anti-LOXL2
(GS‑6624) in NASH and primary sclerosing cholangitis
(NCT01672879104 and NCT01672853,105 respectively).
Targeting multiple stages of the disease process
The ultimate approach is to personalize treatment to
the patient and their disease activity and stage. This
approach might require therapy with a single agent targeting multiple levels of the postulated disease process
in PBC. Is this a realistic goal? Data suggest that the
biological actions of bile acids are substantially more
diverse than had originally been thought, with important proimmune and proinflammatory actions.106 This
finding raises the possibility that the microenvironment
of cholestasis promotes the autoimmune component of
the disease as well as, potentially, generating neoepitopes
that contribute to tolerance breakdown. UDCA, and to
a greater extent the FXR-agonist bile acids, have antiinflammatory and immunomodulatory actions.106 It
is unclear, however, whether the immunomodulatory
capacity of bile acid therapy is sufficient to mitigate
against the seemingly potent cytotoxic CD8 + auto­
reactive T‑cell response directed at BECs throughout the
early stages of PBC.33 Ultimately, a combined approach
of anti-immune and anticholestatic therapy might be
needed to address the combined processes affecting
BECs in PBC.
The issue of the trigger for breakdown of immune
tolerance to PDC and the resulting immune response
targeted against BECs is highly germane to the issue of
the overlap between effective treatment of cholestasis and
immune-mediated injury. If cholestasis is a downstream,
self-sustaining consequence of immune-mediated BEC
injury then it is likely that effective anticholestatic
therapy in isolation will be insufficient to ultimately
prevent ductopenia as that therapy might be insufficient
to prevent the independent immune injury. By contrast,
if autoreactive BEC injury occurs as a consequence of a
primary cholestatic injury causing altered PDC processing 107,108 then early effective targeting of cholestasis might
alter the natural history of immune-mediated BEC injury.
Carefully designed trials of anticholestatic therapy with
appropriate immunological assessment will be essential
to address this question.
Agents with dual efficacy against cholestatic and
fibrotic injury processes seem potentially more realistic. Nor-UDCA has, in addition to its potent choleretic
and cholangiocyte healing actions,109 notable antifibrotic
actions in mouse models of liver disease,110 whilst FXR
agonists also seem to be potently antifibrotic.111 However,
the apparent functional absence of FXR in human (as
opposed to mouse) myofibroblasts argues against this
mechanism of action in humans.112
Future directions in symptomatic therapy
Symptoms of PBC, in particular pruritus and fatigue, are
a major issue for patients. These symptoms are unresponsive to UDCA therapy and no data so far suggest that
second-line prognostic therapies are any more likely to
improve them (indeed obeticholic acid worsens pruritus).113 They are, therefore, important targets for specific
therapies. Although pruritus can be effectively treated
by bile acid sequestrants and rifampicin, such therapy
is frequently poorly tolerated with substantial adverse
effects,114 or lack of efficacy.2 No therapies are currently
proven to improve PBC fatigue.
Targeting pruritus
Pruritus is common and has marked systemic effects,
particularly when accompanied by sleep deprivation.52,115–117 The pruritogens in cholestasis have not been
clearly identified so far, with the result that therapy is
largely empirical (albeit supported by a reasonable trials
evidence base, at least in the case of rifampicin). 118,119
Clinical observations suggest that pruritogens accumulate in the systemic circulation; severe pruritus is at least
in part relieved after treatment with plasmapheresis,
albumin dialysis (for example MARS®, Gambro, Sweden)
or plasma separation and anion absorption.120,121 They
are also secreted into bile—pruritus can be attenuated
after oral administration of the anion exchange resin
cholestyramine,122,123 or in the most severe cases, transiently relieved after nasobiliary drainage.124 Owing to
these observations, trials of ileal apical sodium bile-acid
transporter inhibitors are currently undergoing phase II
evaluation, including LUM001 (NCT01904058125) and
GSK2330672 (NCT01899703126). Pruritogens are also
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VOLUME 12 | MARCH 2015 | 153
REVIEWS
Secreted
into bile
Accumulation
in systemic
circulation
Plasmapheresis
Albumin dialysis
Anion absorption
Cholestyramine
Nasobiliary drainage
Pruritogens
Naltrexone
Sertraline
Rifampicin
Lysophosphatidic
acid
Affect the endogenous
opioidergic and
serotonergic systems
Biotransformed
in liver and/or gut
Autotaxin
Rifampicin
Figure 4 | Therapy for pruritus is largely empirical. Clinical observations suggest
Nature
Reviews
| Gastroenterology
& Hepatology
that potential pruritogens accumulate
in the
systemic
circulation, are secreted
into bile, are biotransformed in the liver and/or gut and affect the endogenous
opioidergic and serotoninergic system. Various treatments target these different
processes. Autotaxin is the enzyme that forms lysophosphatidic acid, a potent
activator of itch neurons. Targeting the autotaxin–lysophosphatidic acid pathway
might provide novel therapy for cholestatic pruritus.
biotransformed in the liver and/or gut (rifampicin is a
potent PXR agonist), and affect the endogenous opioid­
ergic and serotoninergic system—opioid antagonists (for
example, naltrexone) and selective serotonin re­uptake
inhibitors (for example, sertraline) have moderate
a­ntipruritic activity 2,119,127 (Figure 4).
Studies published in the past 3 years indicate that
lysophosphatidic acid, a potent activator of itch neurons,
and autotaxin (also known as ectonucleotide pyrophosphatase/phosphodiesterase 2), the enzyme that catalyses
production of lysophosphatidic acid, might together
represent a key element of the long sought pruritogenic signalling cascade in patients with cholestasis and
itch.116,128,129 Serum levels of autotaxin, but no other pruri­
togen candidate (such as bile salts, endogenous opioids,
histamine, or serotonin) studied so far, correlate with itch
intensity.128,129 Notably, autotaxin serum level mirrors
treatment response for therapeutic interventions such as
colesevelam, rifampicin, nasobiliary drainage, or MARS®
treatment.129 Rifampicin reduced autotaxin transcription in human liver-derived cell lines by PXR-dependent
mechanisms, possibly partly explaining the strong antipruritic effect of rifampicin.129 Targeting the autotaxin and
lysophosphatidic acid pathway offers considerable hope
for novel therapy for PBC and other cholestatic pruritus.
G-protein coupled bile acid receptor 1 (GPBAR1; also
known as takeda G protein-coupled receptor 5 [TGR5]),
might also have an interesting role. GPBAR1 is a G‑proteincoupled receptor that is activated by bile acids and important in the regulation of bile acid homeostasis and the
154 | MARCH 2015 | VOLUME 12
inflammatory response. Mouse models have shown that
bile acids and a Gpabr1-selective agonist activate Gpabr1
on sensory nerves, stimulating the release of neuropeptides
that transmit itch.130,131 Interestingly, high concentrations
of unconjugated deoxycholic acid, a bile acid that is not
or barely found in cholestasis, have been investigated in
these mouse models. The findings might point towards
neurosteroids being important, some of which are better
ligands than most bile acids for GPBAR1. In models of
primary sclerosing cholangitis, activation of GPBAR1 in
BECs promotes bicarbonate secretion, triggers cell proliferation and prevents apoptotic cell death. Pharmacological
activation of Gpbar1 in mice reduces hepatic and systemic
inflammation but also provokes pruritus.132
Targeting fatigue
Chronic fatigue is the most frequently reported symptom
in PBC and markedly affects quality of life.133 Fatigue
does not seem to improve substantially after liver transplantation, which raises the possibility that underlying
processes are at least partially irreversible by the time
advanced disease develops.134 Fatigue in PBC is inherently
complex with both peripheral and central components.
It is strongly associated, in its expression at least, with
sleep disturbance (in particular daytime somnolence)115
and autonomic dysfunction,135 both of which are potential targets for fatigue mitigating approaches. Central
fatigue in PBC is mirrored by fatigue seen, albeit typically at lower levels, in other human cholestatic diseases
such as primary sclerosing cholangitis136 and in animal
models, such as the bile duct ligation rodent in which the
complex behavioural components of fatigue behaviour
seen in patients with PBC (including loss of socialization,
cognitive impairment, reduced activity and frustration)
can be surprisingly effectively modelled.137–140 Human
neurophysiology studies point to abnormalities in regulatory circuits that persist post-transplantation,141 along
with structural white matter lesions and loss of cerebral
autoregulation linked to autonomic dysfunction.142,143
Studies in cholestatic rodents point to direct inflammatory processes linked to monocyte activation and recruitment into the brain, which are directly reversible using
biologic agents.139 Aggressive treatment of inflammatory
pathways, and early aggressive modification of cholestasis
to reduce the inflammatory drive are interesting therapeutic options. Tauroursodeoxcholic acid (TUDCA)
in particular seems to be a potentially attractive agent
because of proven neuroprotective actions through
re­duction of endoplasmic reticulum stress.144
Peripheral fatigue has an overtly energetic element with
increased anaerobic metabolism in muscles seemingly
linked to PDC dysfunction and the anti-PDC antibodies
characteristic of PBC.145 B‑cell depletion therapy to address
this metabolic fatigue in PBC is currently undergoing
evaluation.146 Alternative potentially attractive approaches
include targeting of PDC to normalize low PDC function
(for example using drugs that target pyruvate dehydrogenase kinase, developed for the treatment of type 2 diabetes), and increasing the activity of the monocarboxylase
transporters (in particular MCT4), which are responsible
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for lactate export from muscle, using either exercise
therapy or exercise-mimetic approaches.147,148
Conclusions
The majority of patients with PBC are older (>55 years)4
with mild disease and limited symptoms who respond
well to UDCA (a cheap and well-tolerated therapy). The
treatment challenge in this group is timely diagnosis
and effective delivery of therapy rather than the development of new therapeutics. In the important minority of patients with high risk, treatment unresponsive
or highly symptomatic disease the need for new treatment approaches is now clear and well accepted.149 In the
setting of disease modification, evolution in our understanding of disease mechanisms is evolving rapidly into
the advent of new and re-purposed therapeutics targeting key processes. Excellent opportunities are offered, in
particular, by immunotherapeutic approaches targeting
the upstream immune response postulated to initiate
BEC injury, and by enhanced bile-acid therapies targeting the bile acid biosynthetic and feedback processes
that drive ‘midstream’ injury to BECs. Targeting of the
BEC homeostatic process to enhance recovery following initial injury of the ‘downstream’ fibrotic process are
exciting if more distant potential approaches.
The future challenge in PBC therapeutics will not be
the existence of plausible treatment candidates, but the
need to target therapies at appropriate patients in both
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Acknowledgements
The work of the authors is supported by grant
L001489 from the UK Medical Research Council
(D.E.J.J.). The authors thanks Dr E. Liaskou for her
help in developing Figure 3.
Author contributions
All authors contributed equally to this manuscript.
Supplementary information is linked to the online
version of the paper at www.nature.com/nrgastro.
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